Inhibitors of cellular necrosis

ABSTRACT

The present invention relates to compounds and pharmaceutical preparations and their use in therapy for preventing or treating trauma, ischemia, stroke and degenerative diseases associated with cell death. Methods and compositions of the invention are particularly useful for treating neurological disorders associated with cellular necrosis.

PRIORITY INFORMATION

This application claims the benefit under 35 U.S.C. § 119(e) of thefiling date of U.S. Provisional Application No. 60/498,882 filed Aug.29, 2003, herein incorporated by reference.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under National Instituteof Health Grant No. GM-64703. The Government may have certain rights tothis invention.

FIELD OF INVENTION

The invention relates to compositions and methods for preventing andtreating diseases involving cell death. In particular, the inventionrelates to therapeutic compounds and methods for treating neurologicaldiseases involving cell death.

BACKGROUND OF INVENTION

Acute and chronic neurological diseases can be caused by a number ofdifferent factors. However, many of these diseases are characterized bycell death in specific regions of the central nervous system.

Neurological diseases are a group of maladies that afflict a significantportion of the human population. The medical and socioeconomic impactsof these diseases are significant. Although the etiology of each acuteand chronic neurological disease is likely different, one common featurethat many share is rapid or progressive irreversible cell death inspecific regions of the central nervous system (Standaert, D. G.; Young,A. B. In Goodman & Gilman's The Pharmacological Basis of Therapeutics,Tenth Edition; Hardman, J. G.; Limbird, L. E., Eds.; McGraw-Hill: NewYork, 2001; Chapter 22, pp 549-568; Mattson, M. P. Nature Rev. Mol.Cell. Biol. 2000, 1, 120-129). Compelling evidence is emerging thatneuron cell death occurs in acute neurological diseases, such as strokeand trauma (Raghupathi, R.; Graham, D. I.; McIntosh, T. K. J.Neurotrauma 2000, 17(10), 927-38) and in neurodegenerative diseases,such as Parkinson's disease—PD (Vila, M.; Wu, D. C.; Przedborski, S.Trends in Neuroscience 2001, 24(11), S49-S55), Huntington's disease—HD(McMurry, C. T. Trends in Neuroscience 2001, 24(11), S32-S38),amyotrophic lateral sclerosis—ALS (Beckman, J. S.; Estéves, A. G.; Crow,J. P. Trends in Neuroscience 2001, 24(11), S15-S20), and humanimmunodeficiency virus associated dementia—HAD (Kaul, M.; Garden, G. W.;Lipton, S. A. Nature 2001, 410, 988-994). Studies have also suggestedthat cell death occurs in Alzheimer's disease—AD (Eldadah, B. A.; Faden,A. I. J. Neurotrauma 2000, 17(10), 811-829). Albeit, neurons present inAD may be chronically dysfunctional without necessarily undergoingactive cell death (Selkoe, D. J. Nature 1999, 399 (Suppl), A23-A30).

Most current approaches to developing treatments for neurologicaldiseases, such as stroke, Parkinson's disease (PD), Alzheimer's disease(AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD),and HIV-associated dementia (HAD) target mechanisms that arehypothesized to be involved in the initiation-phase of the disease. Forexample, current approaches involve using compounds that attempt toinhibit the initiation of toxicity caused by aggregation of α-synucleinin PD, or aggregation of β-amyloid, tau, and/or ApoE in AD, oraggregation of huntingtin protein in HD, or oxidative stress fromreactive oxygen species in ALS, or excessive extracellular excitotoxins,such as glutamate, in stroke or trauma. An alternative approach is totarget basic cell death machinery that may be activated as a result of acellular insult. Current approaches are directed towards a specificdeath process called apoptosis involving cysteine proteases calledcaspases. However, a number of recent studies establish that many celldeath paradigms, especially those associated with neurodegeneration,involve non-apoptotic/caspase-independent mechanisms.

Therefore, there is a need in the art for compositions and methods toprevent or treat cellular necrosis including cellular necrosisassociated with neurodegeneration.

SUMMARY OF INVENTION

The present invention provides compounds and pharmaceutical preparationsthat are useful for treating disorders associated with cellularnecrosis.

According to one aspect the invention, the compound has the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents O; Y        represents S; G represents O or NR₇; R₁, R₂, and R₃ represent        independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH,        CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl, amine, piperizine; R₅, R₆, and R₇ represent        independently H or lower alkyl; R₈ represents lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In another aspect the compound has the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents O; Y        represents NR₈; G represents O or NR₇; R₁, R₂, and R₃ represent        independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH,        CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl, amine, piperizine; R₅, R₆, and R₇ represent        independently H or lower alkyl; R₈ represents lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In another aspect of the invention the compound has the formula:

a stereoisomeric form thereof, a pharmaceutically acceptable acid orbase addition salt thereof, wherein X represents O; Y represents NH; R₁,R₂, and R₃ represent independently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈,NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl, substitutedaryl, heteroaryl, or substituted heteroaryl; R₄ represents independentlyH, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl,lower alkyl, substituted lower alkyl, aryl, substituted aryl,heteroaryl, or substituted heteroaryl, amine, piperizine; R₅, R₆ and R₇represent independently H or lower alkyl, except R₆ can not be methyl,ethyl, propyl, isopropyl or t-butyl when R₁, R₂, R₃, R₄, R₅ and R₇ areH; R₈ represents H, lower alkyl, substituted lower alkyl, aryl,substituted aryl, arylalkyl, alkenyl, alkynyl, heteroaryl, orsubstituted heteroaryl; R₉, R₁₀, R₉′, R₁₀′, represent independently H,F, Cl, Br, I, lower alkyl, substituted lower alkyl, or a three to sixmembered cycloalkyl or substituted cycloalkyl that includes C_(n) and/orC_(n)′; n and n′ equals an integer from zero to five.

In another aspect the compound has the formula:

a stereoisomeric form thereof, a pharmaceutically acceptable acid orbase addition salt thereof, wherein X represents O; Y represents NH; R₁,R₂, and R₃ represent independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂,COOH, CO₂R₈, NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,substituted aryl, heteroaryl, or substituted heteroaryl; R₄ representsindependently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈, NO₂,NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted lower alkyl, aryl,substituted aryl, heteroaryl, or substituted heteroaryl, amine,piperizine; R₅, R₆, and R₇ represent independently H or lower alkyl; R₈represents lower alkyl, substituted lower alkyl, aryl, substituted aryl,arylalkyl, alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,R₁₀, R₉, R₁₀′, represent independently H, F, Cl, Br, I, lower alkyl,substituted lower alkyl, or a three to six membered cycloalkyl orsubstituted cycloalkyl that includes C_(n) and/or C_(n)′; n and n′equals an integer from zero to five.

In another aspect the compound has the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents S; Y        represents S; G represents O or NR₇; R₁, R₂, and R₃ represent        independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH,        CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl, amine, piperizine; R₅, R₆, and R₇ represent        independently H or lower alkyl; R₈ represents lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In another aspect of the invention the compound has the formula:

a stereoisomeric form thereof, a pharmaceutically acceptable acid orbase addition salt thereof, wherein X represents S; Y represents NR₈; Grepresents O or NR₇; R₁, R₂, and R₃ represent independently H, OH, OR₈,F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈, NO₂, NHC(O)R₈, lower alkyl,substituted lower alkyl, aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl; R₄ represents independently H, OH, OR₈, F, Cl,Br, I, N(R₈)₂, COOH, CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, loweralkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl, amine, piperizine; R₅, R₆, and R₇ representindependently H or lower alkyl; R₈ represents lower alkyl, substitutedlower alkyl, aryl, substituted aryl, arylalkyl, alkenyl, alkynyl,heteroaryl, or substituted heteroaryl; R₉, R₁₀, R₉′, R₁₀′, representindependently H, F, Cl, Br, I, lower alkyl, substituted lower alkyl, ora three to six membered cycloalkyl or substituted cycloalkyl thatincludes C_(n) and/or C_(n)′; n and n′ equals an integer from zero tofive.

In another aspect of the invention the compound has the formula:

a stereoisomeric form thereof, a pharmaceutically acceptable acid orbase addition salt thereof, wherein X represents S; Y represents NH; Grepresents O or NR₇; R₁, R₂, and R₃ represent independently H, OR₈, F,Cl, Br, I, N(R₈)₂, CO₂R₈, NO₂, NHC(O)R₈, lower alkyl, substituted loweralkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl; R₄represents independently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈, NO₂,NHC(O)R₈, aryl, substituted aryl, heteroaryl, or substituted heteroaryl,amine, piperizine, lower alkyl and substituted lower alkyl except formethyl and methoxyl; R₅, R₆ and R₇ represent independently H or loweralkyl, except R₆ can not be methyl when R₁, R₂, R₃, R₄, R₅ and R₇ are H;R₈ represents H, lower alkyl, substituted lower alkyl, aryl, substitutedaryl, arylalkyl, alkenyl, alkynyl, heteroaryl, or substitutedheteroaryl; R₉, R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I,lower alkyl, substituted lower alkyl, or a three to six memberedcycloalkyl or substituted cycloalkyl that includes C_(n) and/or C_(n)′;n and n equals an integer from zero to five.

In another aspect of the invention the compound has the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents S or O;        Y represents S, NH or NR₈; G represents O or NR₇; R₁, R₂, and R₃        represent independently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈,        NO₂, NHC(O)R₈, aryl, substituted aryl, heteroaryl, or        substituted heteroaryl, amine, piperizine, lower alkyl and        substituted lower alkyl; R₅, R₆ and R₇ represent independently H        or lower alkyl; R₈ represents H, lower alkyl, substituted lower        alkyl, aryl, substituted aryl, arylalkyl, alkenyl, alkynyl,        heteroaryl, or substituted heteroaryl; R₉, R₁₀, R₉′, R₁₀′,        represent independently H, F, Cl, Br, I, lower alkyl,        substituted lower alkyl, or a three to six membered cycloalkyl        or substituted cycloalkyl that includes C_(n) and/or C_(n)′; n        and n′ equals an integer from zero to five.

Another aspect of the invention relates to pharmaceutical preparationscomprising the above compounds and a pharmaceutically acceptablecarrier. In preferred embodiments of the invention the pharmaceuticallyacceptable carrier is chosen from a diluent, a solid filler, and asolvent encapsulating material.

An additional aspect of the invention relates to the use of the abovecompounds for treating necrotic cell diseases including trauma,ischemia, stroke, cardiac infarction, infection and sepsis. In preferredembodiment the necrotic cell disease is a neurodegenerative disease suchas Parkinson's disease, Alzheimer's disease, amyotrophic lateralsclerosis, Huntington's disease, and HIV-associated dementia.

An additional aspect of the invention relates to the use of acombination of two or more compounds that inhibit cellular necrosis(e.g., heterocylcic thiohydantoin, hydantoin, oxazolidinone,thioxo-oxazolidinone, pyrimidinone, oxazinanone compounds, orcombinations thereof) according to a treatment method of the invention.

An additional aspect of the invention relates to the use of one or moreof the aforementioned compounds in combination with one or moreadditional compounds or agents such as those described herein. Inpreferred embodiments of the invention the additional compound(s) is(are) selected from apoptosis inhibitors, PARP inhibitors, Srcinhibitors, agents for treating cardiovascular disorders andanti-microbial agents.

An additional aspect of the invention relates to processes for producingone or more heterocyclic compounds comprising a thiohydantoin,hydantoin, oxazolidinone, thioxo-oxazolidinone, pyrimidinone, oroxazinanone moiety.

A further aspect of the present invention relates to the synthesis ofcombinatorial libraries of the heterocyclic compounds comprising athiohydantoin, hydantoin, oxazolidinone, thioxo-oxazolidinone,pyrimidinone, or oxazinanone moiety, and the screening of thoselibraries for biological activity, e.g. in assays based on cell death(apoptosis, necrosis, or a combination of both) and in animal models ofdisease including trauma, ischemia (e.g. stroke, myocardial infractionand the like), and neurodegenerative diseases such as Parkinson'sdisease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis(ALS), Huntington's disease (HD), infectious encephalopathies, dementia,HIV-associated dementia (HAD), etc.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a graph bar that shows the effects of anti-necrotic compound893-01.

FIG. 2 depicts a general scheme for synthesizing a heterocyclic compoundcomprising a thiohydantoin or hydantoin moiety.

FIG. 3 depicts a general scheme for synthesizing a heterocyclic compoundcomprising a thiohydantoin moiety.

FIG. 4 depicts a general scheme for synthesizing a heterocyclic compoundcomprising a hydantoin moiety.

FIG. 5 depicts a scheme for the preparation of indoles.

FIG. 6 shows a scheme for the preparation of 7-oxygenated indoles.

FIG. 7 shows a scheme for the preparation of7-(2-methoxy-ethoxy)-1H-indole.

FIG. 8 depicts (A) a general scheme for enantioselectively synthesizinga heterocyclic compound comprising a hydantoin moiety (B) scheme forsynthesizing compounds 893-31 and 893-32.

FIG. 9 depicts a general scheme for synthesizing hydantoins.

FIG. 10 depicts a general scheme for the preparation of oxazolidinones.

FIG. 11 is a graph that shows the cytotoxicity of hydantoin andthiohydantoin compounds.

DETAILED DESCRIPTION

The invention provides compounds that prevent cell death and are usefulas therapeutic agents for treating subjects afflicted with necrotic celldisease, such as trauma, ischemic and neurological diseases, andparticularly neurodegenerative diseases. Compounds of the invention arealso useful for understanding the patho-physiology of these diseases.

Compounds of the invention are low molecular weight molecules of theformula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein    -   X represents O or S;    -   Y represents S, NH, or NR₈;    -   G represents O or NR₇;    -   R₁, R₂, and R₃ represent independently H, OH, OR₈, F, Cl, Br, I,        N(R₈)₂, COOH, CO₂R₈, NO₂, NHC(O)R₈, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl;    -   R₄ represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂,        COOH, CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl,        substituted lower alkyl, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, amine, or piperizine, but preferably Cl,        F or methoxyl;    -   R₅, R₆, and R₇ represent independently H or lower alkyl;    -   R₈ represents lower alkyl, substituted lower alkyl, aryl,        substituted aryl, arylalkyl, alkenyl, alkynyl, heteroaryl, or        substituted heteroaryl;    -   R₉, R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I,        lower alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl the includes C_(n) and/or        C_(n)′;    -   n and n′ equals an integer from zero to five;    -   with restrictions on the following alternative embodiments:    -   i) when X represents O, Y represents NH and G represents NR₇, R₆        can not be a methyl, ethyl, propyl, isopropyl or t-butyl when        R₁, R₂, R₃, R₄, R₅ and R₇ represent H;    -   ii) when X represents S, Y represents NH and G represents NR₇,        R₆ can not be a methyl when R₁, R₂, R₃, R₄, R₅ and R₇ represent        H;    -   iii) and when X represents S, Y represents NH and G represents        NR₇, R₄ can not be a methyl or methoxyl.

In another aspect of the invention compounds of the invention are lowmolecular weight molecules of the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein    -   X represents O or S;    -   Y represents S, NH, or NR₈;    -   G represents O or NR₇;    -   R₁, R₂, and R₃ represent independently H, OH, OR₈, F, Cl, Br, I,        N(R₈)₂, COOH, CO₂R₈, NO₂, NHC(O)R₈, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl;    -   R₄ represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂,        COOH, CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl,        substituted lower alkyl, aryl, substituted aryl, heteroaryl,        substituted heteroaryl, amine, or piperizine, but preferably Cl        or F;    -   R₅, R₆, and R₇ represent independently H or lower alkyl;    -   R₈ represents lower alkyl, substituted lower alkyl, aryl,        substituted aryl, arylalkyl, alkenyl, alkynyl, heteroaryl, or        substituted heteroaryl;    -   R₉, R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I,        lower alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl the includes C_(n) and/or        C_(n)′; and wherein n and n′ equals an integer from zero to        five.

In one aspect, compounds of the invention inhibit necrotic cell death byinhibiting caspase-independent mechanisms that are activated after acell-death initiating event. According to the invention, the inhibitionof caspase-independent cell-death mechanisms provides severaladvantages, including a high therapeutic efficacy and a broad utilityagainst many diseases, including but not limited to trauma, ischemia andneurological diseases. In addition, compounds of the invention can beused in assays for novel molecular targets that are associated withcaspase-independent induced cell death.

Cells die in two morphologically distinct ways (Wyllie, A. H.; Kerr, J.F. R.; Currie, A. R. Int. Rev. Cytol. 1980, 68, 251-306). One of theseprocesses, called apoptosis, is characterized by a number of conservedand highly regulated steps including concurrent nucleus and cytoplasmcondensation (although cytoplasmic organelles initially remain intact),DNA degradation, membrane blebbing and caspase-mediated cleavage ofvarious cellular factors. Apoptosis culminates in cellular fragmentationinto apoptotic bodies, which are phagocytosed by adjacent cells,including macrophages. Therefore, this process does not lead to aninflammatory response. Apoptosis is a genetically regulated process(Yuan, J.; Yankner, B. A. Nature 2000, 407, 802-809; Cryns, V.; Yuan, J.Genes & Develop. 1998, 12, 1551-1570) necessary during both developmentand for maintaining an organism's homeostasis. However, in certainpathological conditions this process, which would normally besuppressed, is initiated leading to cell death and dysfunction. Many keycellular targets in this cascade have been identified and some serve aspotential targets for therapeutic intervention. For example, one familyof enzymes discovered to play an integral role in this process iscaspases, which are cysteine proteases (Talanian, R. V.; Brady, K. D.;Cryns, V. L. J. Med. Chem. 2000, 43(18), 3351-3371).

A second, morphologically distinct way that cells die, called necrosis(Syntichaki, P.; Tavernarakis, N. EMBO Rep. 2002, 3(7), 604-609), ischaracterized by cell membrane and organelle disruption, cell swelling,mitochondria impairment, followed by cell lyses (Martin, L. J.,Al-Abdulla, N. A.; Brambrink, A. M.; Kirsch, J. R.; Sieber, F. E.;Portera-Cailliau, C. Brain Res. Bull. 1998, 46(4), 281-309).Condensation of chromatin occurs, but only with diffuse irregular shapedmasses being formed. Also, cell lyses typically are accompanied by aninflammatory response. Although the underlying biochemical events inthis process are not well understood, necrotic cell death is known toplay a very prominent role in many pathological conditions, especiallyduring neurodegeneration (Nicotera, P., Leist, M.; Ferrando-May E.Biochem. Soc. Symp. 1999, 66, 69-73). It is thought that inhibition ofapoptosis often does not completely block cell death, but rather resultsin a cell switching from an apoptotic to a necrotic mechanism.Therefore, identifying and preparing low molecular weight molecules thatprevent necrotic cell death can assist in the basic understanding ofthis process and provide useful compounds for therapeutic intervention.Compounds of the invention target important aspects of neurodegenerationnot addressed by current strategies.

According to the invention, necrosis can be associated with a conditionincluding, but not limited to, an infection, a toxin, a poison,radiation, physical trauma, inflammation, a lack of nutrient or oxygensupply, a chemical imbalance, an interruption of blood supply, otherconditions leading to cell or tissue death, or a combination of two ormore of the above. For example, cell or tissue necrosis can beassociated with any one or more of the following conditions: an abscess,ague, anemia, ankylosis, anoxia, apnea, arthritis, asphyxiation, asthma,ataxia, atrophy, backache, bleeding, blennorhea, cachexia, caries,colic, constipation, convulsion, coughing, cyanosis, diarrhea,dizziness, dropsy, dry gangrene, dysentery, dyspepsia, dyspnea, edema,emaciation, fainting, fatigue, fever, fibrillation, gas gangrene,genetic diseases, high blood pressure, hydrops, hypertension,hypotension, icterus, indigestion, inflammation, insomnia, itching,jaundice, low blood pressure, lumbago, marasmus, moist gangrene, noma,pain, paralysis, pruritus, rash, rheum, sclerosis, seizure, shock, skineruption, sore, spasm, sphacelation, tabes, tachycardia, tooth decay,tumor, upset stomach, vertigo, vomiting, or wasting.

Accordingly, necrosis can be localized to a group of living cells or canbe spread over one or more larger tissue areas. In some embodiments,necrosis can be associated with gangrene, sphacelus, ischemic necrosis,avascular necrosis (e.g., of the bone), meningitis, and other conditionsincluding but not limited to those described herein.

The term “necrotic cell disease” refers to acute diseases including butnot limited to trauma, ischemia, stroke, cardiac infarction, anthraxlethal toxin induced septic shock, sepsis, cell death induced by LPS,and HIV induced T-cell death leading to immunodeficiency. The term“necrotic cell disease” also includes but is not limited to chronicneurodegenerative diseases, such as Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, Alzheimer's disease, infectiousencelopathies, dementia such as HIV associated dementia.

The invention is based, in part, on the discovery that a series ofcompounds including, but not limited to, the following:

-   -   act as inhibitors of cellular necrosis. According to the        invention, these compounds, and certain derivatives thereof, are        useful to treat diseases such as trauma, ischemia (e.g. stroke,        myocardial infarction and the like), and neurodegenerative        diseases. Useful thiohydantoin, hydantoin, oxazolidinone,        thioxo-oxazolidinone, pyrimidinone, or oxazinanone compound        derivatives preferably have small substituents at the 7 position        of the indole ring such as halogen, methyl, and methoxyl and        groups such as methyl and other lower alkyl groups at the        (thio)hydantoin imide nitrogen.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 12 or fewer carbon atoms inits backbone (e.g., C₁-C₁₂ for straight chain, C₃-C₁₂ for branchedchain), and more preferably 6 or fewer, and even more preferably 4 orfewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms intheir ring structure, and more preferably have 5, 6 or 7 carbons in thering structure.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure, and even more preferably from one to four carbonatoms in its backbone structure. Likewise, “lower alkenyl” and “loweralkynyl” have similar chain lengths. Preferred alkyl groups are loweralkyls. In preferred embodiments, a substituent designated herein asalkyl is a lower alkyl.

As used herein, the term “halogen” designates —F, —Cl, —Br or —I; theterm “sulfhydryl” means —SH; and the term “hydroxyl” means —OH.

The term “methyl” refers to the monovalent radical —CH₃, and the term“methoxyl” refers to the monovalent radical —CH₂OH.

The term “aralkyl” or “arylalkyl”, as used herein, refers to an alkylgroup substituted with an aryl group (e.g., an aromatic orheteroaromatic group).

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond respectively.

The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstitutedbenzenes, respectively. For example, the names 1,2-dimethylbenzene andortho-dimethylbenzene are synonymous.

The terms “heterocyclyl” or “heterocyclic group” or “heteroaryl” referto 3- to 10-membered ring structures, more preferably 3- to 7-memberedrings, whose ring structures include one to four heteroatoms.Heterocycles can also be polycycles. Heterocyclyl groups include, forexample, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene,xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole,isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine,isoindole, indole, indazole, purine, quinolizine, isoquinoline,quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline,cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine,pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine,furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole,piperidine, piperazine, morpholine, lactones, lactams such asazetidinones and pyrrolidinones, sultams, sultones, and the like. Theheterocyclic ring can be substituted at one or more positions with suchsubstituents as described above, as for example, halogen, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl,silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester,heterocyclyl, aromatic or heteroaromatic moiety, —CF₃, —CN, or the like.

As used herein, the definition of each expression, e.g. alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl,cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moiety, —CF₃, —CN, or the like. For purposes of thisinvention, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms. Thisinvention is not intended to be limited in any manner by the permissiblesubstituents of organic compounds.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.In certain embodiments, the present invention relates to a compoundrepresented by any of the structures outlined herein, wherein thecompound is a single stereoisomer.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., functioning as inhibitors of cellularnecrosis), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds of the present invention may be prepared by themethods illustrated in the general reaction schemes as, for example,described below, or by modifications thereof, using readily availablestarting materials, reagents and conventional synthesis procedures. Inthese reactions, it is also possible to make use of variants, which arein themselves known, but are not mentioned here.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

Accordingly, in certain embodiments, the invention provides a compoundof the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents O; Y        represents S; G represents O or NR₇; R₁, R₂, and R₃ represent        independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH,        CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl, amine, piperizine; R₅, R₆, and R₇ represent        independently H or lower alkyl; R₈ represents lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In certain embodiments the invention provides a compound of the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents O; Y        represents NR₈; G represents O or NR₇; R₁, R₂, and R₃ represent        independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH,        CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl, amine, piperizine; R₅, R₆, and R₇ represent        independently H or lower alkyl; R₈ represents lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In certain embodiments the invention provides a compound of the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents O; Y        represents NH; R₁, R₂, and R₃ represent independently H, OR₈, F,        Cl, Br, I, N(R₈)₂, CO₂R₈, NO₂, NHC(O)R₈, lower alkyl,        substituted lower alkyl, aryl, substituted aryl, heteroaryl, or        substituted heteroaryl; R₄ represents independently H, OR₈, F,        Cl, Br, I, N(R₈)₂, CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower        alkyl, substituted lower alkyl, aryl, substituted aryl,        heteroaryl, or substituted heteroaryl, amine, piperizine; R₅, R₆        and R₇ represent independently H or lower alkyl, except R₆ can        not be methyl, ethyl, propyl, isopropyl or t-butyl when R₁, R₂,        R₃, R₄, R₅ and R₇ are H; R₈ represents H, lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In certain embodiments the invention provides a compound of the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents O; Y        represents NH; R₁, R₂, and R₃ represent independently H, OH,        OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈, NO₂, NHC(O)R₈, lower        alkyl, substituted lower alkyl, aryl, substituted aryl,        heteroaryl, or substituted heteroaryl; R₄ represents        independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈,        NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted lower        alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl, amine, piperizine; R₅, R₆, and R₇ represent        independently H or lower alkyl; R₈ represents lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In certain embodiments the invention provides a compound of the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents S; Y        represents S; G represents O or NR₇; R₁, R₂, and R₃ represent        independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH,        CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl, amine, piperizine; R₅, R₆, and R₇ represent        independently H or lower alkyl; R₈ represents lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In certain embodiments the invention provides a compound of the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents S; Y        represents NR₈; G represents O or NR₇; R₁, R₂, and R₃ represent        independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH,        CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, lower alkyl, substituted        lower alkyl, aryl, substituted aryl, heteroaryl, or substituted        heteroaryl, amine, piperizine; R₅, R₆, and R₇ represent        independently H or lower alkyl; R₈ represents lower alkyl,        substituted lower alkyl, aryl, substituted aryl, arylalkyl,        alkenyl, alkynyl, heteroaryl, or substituted heteroaryl; R₉,        R₁₀, R₉′, R₁₀′, represent independently H, F, Cl, Br, I, lower        alkyl, substituted lower alkyl, or a three to six membered        cycloalkyl or substituted cycloalkyl that includes C_(n) and/or        C_(n)′; n and n′ equals an integer from zero to five.

In certain embodiments the invention provides a compound of the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents S; Y        represents NH; G represents O or NR₇; R₁, R₂, and R₃ represent        independently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈, NO₂,        NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈,        NO₂, NHC(O)R₈, aryl, substituted aryl, heteroaryl, or        substituted heteroaryl, amine, piperizine, lower alkyl and        substituted lower alkyl except for methyl and methoxyl; R₅, R₆        and R₇ represent independently H or lower alkyl, except R₆ can        not be methyl when R₁, R₂, R₃, R₄, R₅ and R₇ are H; R₈        represents H, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, arylalkyl, alkenyl, alkynyl, heteroaryl, or        substituted heteroaryl; R₉, R₁₀, R₉′, R₁₀′, represent        independently H, F, Cl, Br, I, lower alkyl, substituted lower        alkyl, or a three to six membered cycloalkyl or substituted        cycloalkyl that includes C_(n) and/or C_(n)′; n and n′ equals an        integer from zero to five.

In certain embodiments the invention provides a compound of the formula:

-   -   a stereoisomeric form thereof, a pharmaceutically acceptable        acid or base addition salt thereof, wherein X represents S or O;        Y represents S, NH, or NR₈; G represents O or NR₇; R₁, R₂, and        R₃ represent independently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈,        NO₂, NHC(O)R₈, lower alkyl, substituted lower alkyl, aryl,        substituted aryl, heteroaryl, or substituted heteroaryl; R₄        represents independently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈,        NO₂, NHC(O)R₈, aryl, substituted aryl, heteroaryl, or        substituted heteroaryl, amine, piperizine, lower alkyl and        substituted lower alkyl except for methyl and methoxyl; R₅, R₆        and R₇ represent independently H or lower alkyl; R₈ represents        H, lower alkyl, substituted lower alkyl, aryl, substituted aryl,        arylalkyl, alkenyl, alkynyl, heteroaryl, or substituted        heteroaryl; R₉, R₁₀, R₉′, R₁₀′, represent independently H, F,        Cl, Br, I, lower alkyl, substituted lower alkyl, or a three to        six membered cycloalkyl or substituted cycloalkyl that includes        C_(n) and/or C_(n)′; n and n′ equals an integer from zero to        five.

In another aspect, the present invention provides pharmaceuticallyacceptable compositions, which comprise a therapeutically effectiveamount of one or more of the compounds described herein, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. As described in detail, the pharmaceuticalcompositions of the present invention may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, boluses, powders, granules,pastes for application to the tongue; parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; topical application, for example, as acream, ointment, or a controlled-release patch or spray applied to theskin; intravaginally or intrarectally, for example, as a pessary, creamor foam; sublingually; ocularly; transdermally; or nasally, pulmonaryand to other mucosal surfaces.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations. Examples of such formulations include, but are not limitedto DMSO, 10 mM DMSO, 8% hydroxypropyl-beta-cyclodextrin in PBS,propylene glycol, etc.

For example, in a certain embodiment the compounds of the invention,such as 893-54 can be used as 4 mM solution in 8%hydroxypropyl-beta-cyclodextrin in PBS for parenteral administration. Inanother embodiment 893-54 (0.40 mg/mL) was formulated as follows: 5.975mg of 893-54 was weighed into a pre-cleaned 20 mL glass vial, 1.49 mL ofpropylene glycol was added to the vial followed by vortexing andsonication for 15 minutes, and 13.41 mL of sterile normal saline wasadded for a total volume of 14.9 mL followed by vortexing.

As set out herein, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and are,thus, capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable acids. The term “pharmaceutically-acceptablesalts” in this respect refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the present invention. Thesesalts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedcompound of the invention in its free base form with a suitable organicor inorganic acid, and isolating the salt thus formed during subsequentpurification. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19.)

The pharmaceutically acceptable salts of the subject compounds includethe conventional nontoxic salts or quaternary ammonium salts of thecompounds, e.g., from non-toxic organic or inorganic acids. For example,such conventional nontoxic salts include those derived from inorganicacids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. The amount of active ingredient which canbe combined with a carrier material to produce a single dosage form willvary depending upon the host being treated, the particular mode ofadministration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the compound which produces a therapeutic effect.Generally, this amount will range from about 1% to about 99% of activeingredient, preferably from about 5% to about 70%, most preferably fromabout 10% to about 30%.

In certain embodiments, a formulation of the present invention comprisesan excipient selected from the group consisting of cyclodextrins,liposomes, micelle forming agents, e.g., bile acids, and polymericcarriers, e.g., polyesters and polyanhydrides; and a compound of thepresent invention. In certain embodiments, an aforementioned formulationrenders orally bioavailable a compound of the present invention.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound of the present invention withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound of the present invention withliquid carriers, or finely divided solid carriers, or both, and then, ifnecessary, shaping the product.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol, glycerol monostearate, and non-ionic surfactants;absorbents, such as kaolin and bentonite clay; lubricants, such a talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, and mixtures thereof; and coloring agents. In the caseof capsules, tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-shelled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made in asuitable machine in which a mixture of the powdered compound ismoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be formulated for rapid release,e.g., freeze-dried. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions that can be dissolvedin sterile water, or some other sterile injectable medium immediatelybefore use. These compositions may also optionally contain opacifyingagents and may be of a composition that they release the activeingredient(s) only, or preferentially, in a certain portion of thegastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients, such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the present invention to the body. Dissolvingor dispersing the compound in the proper medium can make such dosageforms. Absorption enhancers can also be used to increase the flux of thecompound across the skin. Either providing a rate controlling membraneor dispersing the compound in a polymer matrix or gel can control therate of such flux.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more compounds of the invention incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, antioxidants, buffers, bacteriostats, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenyl sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

In another aspect, the present invention relates to a method of treatinga disease associated with cellular necrosis. In particular, theinvention provides methods for preventing or treating a disorderassociated with cellular necrosis in a mammal, comprising the step ofadministering to said mammal a therapeutically effective amount of acompound or therapeutic preparation of the present invention. In certainembodiments, the disorder associated with cellular necrosis is aneurological disorder such as trauma, ischemia or stroke. In otherembodiments, the neurological disorder is a neurodegenerative disease,such as Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophiclateral sclerosis (ALS), Huntington's disease (HD), and HIV-associateddementia (HAD). In other embodiments the disorder is an ischemic diseaseof organs including but not limited to brain, heart, kidney, and liver.In certain embodiments, the mammal is a primate, canine or felinesubject. In other embodiments, the mammal is a human subject.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment. Atherapeutically effective amount for treating a neurological disorder isan amount sufficient to inhibit necrosis in at least a subset of cellsthat were exposed to a cell-death initiating event. Accordingly, atherapeutically effective amount prevents or minimizes diseaseprogression associated with cellular necrosis. Disease progression canbe monitored relative to an expected disease progression that is basedon population studies, controlled observations in individuals, or acombination of both.

In certain embodiments, a compound or pharmaceutical preparation isadministered orally. In other embodiments, the compound orpharmaceutical preparation is administered intravenously. Alternativeroutes of administration include sublingual, intramuscular, andtransdermal administrations.

In certain embodiments, the present invention relates to ligands forinhibiting cell death, wherein the ligands are represented by any of thestructures outlined above, and any sets of definitions associated withone of those structures. In certain embodiments, the ligands of thepresent invention are inhibitors of cell death. In any event, theligands of the present invention preferably exert their effect oninhibiting cell death at a concentration less than about 50 micromolar,more preferably at a concentration less than about 10 micromolar, andmost preferably at a concentration less than 1 micromolar.

The compounds of the invention can be tested in standard animal modelsof stroke and standard protocols such as described by Hara, H., et al.Proc Natl Acad Sci U S A, 1997. 94(5): 2007-12.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1% to 99.5% (morepreferably, 0.5% to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracistemally and topically, as by powders, ointments ordrops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically-acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts. A daily,weekly, or monthly dosage (or other time interval) can be used.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required to achievethe desired therapeutic effect and then gradually increasing the dosageuntil the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally doses of the compounds of thisinvention for a patient, when used for the indicated effects, will rangefrom about 0.0001 to about 100 mg per kg of body weight per day.Preferably the daily dosage will range from 0.001 to 50 mg of compoundper kg of body weight, and even more preferably from 0.01 to 10 mg ofcompound per kg of body weight.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, it is preferable to administer the compound as apharmaceutical formulation (composition).

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of one or more of the subject compounds, as described above,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: oral administration, for example, drenches(aqueous or non-aqueous solutions or suspensions), tablets, boluses,powders, granules, pastes for application to the tongue; parenteraladministration, for example, by subcutaneous, intramuscular orintravenous injection as, for example, a sterile solution or suspension;topical application, for example, as a cream, ointment or spray appliedto the skin, lungs, or oral cavity; or intravaginally or intravectally,for example, as a pessary, cream or foam; sublingually; ocularly;transdermally; nasally; pulmonary or to other mucosal surfaces.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure. The patient receiving this treatment is any animal in need ofsuch treatment, including primates, in particular humans, and othermammals such as equines, cattle, swine and sheep; and poultry and petsin general.

In another aspect of the invention the compounds can be administered incombination with compounds that are apoptosis inhibitors. The term“apoptosis inhibitor” refers to compounds that inhibit apoptosis,including but not limited to reversible and irreversible caspaseinhibitors. An example of an apoptosis inhibitor includes zVAD(N-benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone), IETD(N-acetyl-Ile-Glu-Thr-Asp-al), YVAD(N-benzyloxycarbonyl-Tyr-Val-Ala-Asp-(OMe) fluoromethyl ketone), DEVD(N-[2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoyl]-L-α-aspartyl-L-α-glutamyl-N-[(1S)-1-(carboxymethyl)-3-fluoro-2-oxopropyl]-L-Valinamide),and LEHD (N-acetyl-Leu-Glu-His-Asp-al).

In some preferred embodiments the compounds of the invention areadministered in combination with PARP poly(ADP-ribose) polymeraseinhibitors. Non-limiting examples of PARP inhibitors include6(5H)-Phenanthridinone, 4-Amino-1,8-naphthalimide, 1,5-Isoquinolinediol,and 3-Aminobenzamide.

In yet other preferred embodiments the compounds of the invention areadministered in combination with Src inhibitors. Src proteins aremammalian cytoplasmic tyrosine kinases that play an extensive role insignal transduction. Examples of Src inhibitors include but are notlimited to:PP1(1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine),PP2(3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine),damnacanthal (3-hydroxy-1-methoxy-2-anthraquinonecarboxaldehyde), andSU-5565.

The term “trauma” as used herein refers to any physical damage to thebody caused by violence, accident, fracture etc. The term “ischemia”refers to a cardiovascular disorder characterized by a low oxygen stateusually due to the obstruction of the arterial blood supply orinadequate blood flow leading to hypoxia in the tissue. The term“stroke” refers to cardiovascular disorders caused by a blood clot orbleeding in the brain, most commonly caused by an interruption in theflow of blood in the brain as from clot blocking a blood vessel, and incertain embodiments of the invention the term stroke refers to ischemicstroke or hemorrhagic stroke. The term “myocardial infarction” refers toa cardiovascular disorder characterized by localized necrosis resultingfrom obstruction of the blood supply.

The methods of the invention involve, in some aspects, combinations ofcompounds that are inhibitors of cellular necrosis (e.g., heterocyclicthiohydantoin, hydantoin, oxazolidinone, thioxo-oxazolidinone,pyrimidinone, or oxazinanone compounds, or combinations thereof) withagents for the treatment of cardiovascular disorders. The term “agentsfor treating cardiovascular disorders” include compounds selected fromthe group consisting of anti-inflammatory agents, anti-thromboticagents, anti-platelet agents, fibrinolytic agents, lipid reducingagents, direct thrombin inhibitors, glycoprotein II b/IIIa receptorinhibitors, agents that bind to cellular adhesion molecules and inhibitthe ability of white blood cells to attach to such molecules (e.g.anti-cellular adhesion molecule antibodies), calcium channel blockers,beta-adrenergic receptor blockers, cyclooxygenase-2 inhibitors,angiotensin system inhibitors, and/or any combinations thereof.

One preferred agent is aspirin.

“Anti-inflammatory” agents include Alclofenac; AlclometasoneDipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide;Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac;Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen;Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide;Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate;Clobetasone Butyrate; Clopirac; Cloticasone Propionate; CormethasoneAcetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone;Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium;Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate;Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab;Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac;Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate;Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lomoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone;Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam;Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; TolmetinSodium; Triclonide; Triflumidate; Zidometacin; Glucocorticoids;Zomepirac Sodium.

“Anti-thrombotic” and/or “fibrinolytic” agents include Plasminogen (toplasmin via interactions of prekallikrein, kininogens, Factors XII,XIIIa, plasminogen proactivator, and tissue plasminogen activator[TPA])Streptokinase; Urokinase: Anisoylated Plasminogen-StreptokinaseActivator Complex; Pro-Urokinase; (Pro-UK); rTPA (alteplase or activase;r denotes recombinant); rPro-UK; Abbokinase; Eminase; SreptaseAnagrelide Hydrochloride; Bivalirudin; Dalteparin Sodium; DanaparoidSodium; Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin Sodium;Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; retaplase; Trifenagrel;Warfarin; Dextrans.

“Anti-platelet” agents include Clopridogrel; Sulfinpyrazone; Aspirin;Dipyridamole; Clofibrate; Pyridinol Carbamate; PGE; Glucagon;Antiserotonin drugs; Caffeine; Theophyllin Pentoxifyllin; Ticlopidine;Anagrelide.

“Lipid reducing” agents include gemfibrozil, cholystyramine, colestipol,nicotinic acid, probucol lovastatin, fluvastatin, simvastatin,atorvastatin, pravastatin, cirivastatin.

“Direct thrombin inhibitors” include hirudin, hirugen, hirulog,agatroban, PPACK, thrombin aptamers.

“Glycoprotein IIb/IIIa receptor inhibitors” are both antibodies andnon-antibodies, and include but are not limited to ReoPro (abcixamab),lamifiban, tirofiban.

“Calcium channel blockers” are a chemically diverse class of compoundshaving important therapeutic value in the control of a variety ofdiseases including several cardiovascular disorders, such ashypertension, angina, and cardiac arrhythmias (Fleckenstein, Cir. Res.v. 52, (suppl. 1), p. 13-16 (1983); Fleckenstein, Experimental Facts andTherapeutic Prospects, John Wiley, New York (1983); McCall, D., CurrPract Cardiol, v. 10, p. 1-11 (1985)). Calcium channel blockers are aheterogenous group of drugs that prevent or slow the entry of calciuminto cells by regulating cellular calcium channels. (Remington, TheScience and Practice of pharmacy, Nineteenth Edition, Mack PublishingCompany, Eaton, Pa., p. 963 (1995)). Most of the currently availablecalcium channel blockers, and useful according to the present invention,belong to one of three major chemical groups of drugs, thedihydropyridines, such as nifedipine, the phenyl alkyl amines, such asverapamil, and the benzothiazepines, such as diltiazem. Other calciumchannel blockers useful according to the invention, include, but are notlimited to, amrinone, amlodipine, bencyclane, felodipine, fendiline,flunarizine, isradipine, nicardipine, nimodipine, perhexylene,gallopamil, tiapamil and tiapamil analogues (such as 1993RO-11-2933),phenyloin, barbiturates, and the peptides dynorphin, omega-conotoxin,and omega-agatoxin, and the like and/or pharmaceutically acceptablesalts thereof.

“Beta-adrenergic receptor blocking agents” are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,hedroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy)phthalide.The above-identified compounds can be used as isomeric mixtures, or intheir respective levorotating or dextrorotating form.

Cyclooxygenase-2 (COX-2) is a recently identified new form of acyclooxygenase. “Cyclooxygenase” is an enzyme complex present in mosttissues that produces various prostaglandins and thromboxanes fromarachidonic acid. Non-steroidal, anti-inflammatory drugs exert most oftheir anti-inflammatory, analgesic and antipyretic activity and inhibithormone-induced uterine contractions and certain types of cancer growththrough inhibition of the cyclooxygenase (also known as prostaglandinG/H synthase and/or prostaglandin-endoperoxide synthase). Initially,only one form of cyclooxygenase was known, the “constitutive enzyme” orcyclooxygenase-1 (COX-1). It and was originally identified in bovineseminal vesicles.

Cyclooxygenase-2 (COX-2) has been cloned, sequenced and characterizedinitially from chicken, murine and human sources (See, e.g., U.S. Pat.No. 5,543,297, issued Aug. 6, 1996 to Cromlish, et al., and assigned toMerck Frosst Canada, Inc., Kirkland, Calif., entitled: “Humancyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity”). This enzyme is distinct from the COX-1. COX-2, is rapidlyand readily inducible by a number of agents including mitogens,endotoxin, hormones, cytokines and growth factors. As prostaglandinshave both physiological and pathological roles, it is believed that theconstitutive enzyme, COX-1, is responsible, in large part, forendogenous basal release of prostaglandins and hence is important intheir physiological functions such as the maintenance ofgastrointestinal integrity and renal blood flow. By contrast, it isbelieved that the inducible form, COX-2, is mainly responsible for thepathological effects of prostaglandins where rapid induction of theenzyme would occur in response to such agents as inflammatory agents,hormones, growth factors, and cytokines. Therefore, it is believed thata selective inhibitor of COX-2 has similar anti-inflammatory,antipyretic and analgesic properties to a conventional non-steroidalanti-inflammatory drug, and in addition inhibits hormone-induced uterinecontractions and also has potential anti-cancer effects, but withreduced side effects. In particular, such COX-2 inhibitors are believedto have a reduced potential for gastrointestinal toxicity, a reducedpotential for renal side effects, a reduced effect on bleeding times andpossibly a decreased potential to induce asthma attacks inaspirin-sensitive asthmatic subjects, and are therefore useful accordingto the present invention.

A number of selective “COX-2 inhibitors” are known in the art. Theseinclude, but are not limited to, COX-2 inhibitors described in U.S. Pat.No. 5,474,995 “Phenyl heterocycles as cox-2 inhibitors”; U.S. Pat. No.5,521,213 “Diaryl bicyclic heterocycles as inhibitors ofcyclooxygenase-2”; U.S. Pat. No. 5,536,752 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,550,142 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,552,422 “Aryl substituted 5,5 fusedaromatic nitrogen compounds as anti-inflammatory agents”; U.S. Pat. No.5,604,253 “N-benzylindol-3-yl propanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,604,260“5-methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2”;U.S. Pat. No. 5,639,780 N-benzyl indol-3-yl butanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,677,318Diphenyl-1,2-3-thiadiazoles as anti-inflammatory agents”; U.S. Pat. No.5,691,374 “Diaryl-5-oxygenated-2-(5H)-furanones as COX-2 inhibitors”;U.S. Pat. No. 5,698,584 “3,4-diaryl-2-hydroxy-2,5-dihydrofurans asprodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,710,140 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,733,909 “Diphenylstilbenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,789,413“Alkylated styrenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No.5,817,700 “Bisaryl cyclobutenes derivatives as cyclooxygenaseinhibitors”; U.S. Pat. No. 5,849,943 “Stilbene derivatives useful ascyclooxygenase-2 inhibitors”; U.S. Pat. No. 5,861,419 “Substitutedpyridines as selective cyclooxygenase-2 inhibitors”; U.S. Pat. No.5,922,742 “Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2inhibitors”; U.S. Pat. No. 5,925,631 “Alkylated styrenes as prodrugs toCOX-2 inhibitors”; all of which are commonly assigned to Merck FrosstCanada, Inc. (Kirkland, Calif.). Additional COX-2 inhibitors are alsodescribed in U.S. Pat. No. 5,643,933, assigned to G. D. Searle & Co.(Skokie, Ill.), entitled: “Substituted sulfonylphenylheterocycles ascyclooxygenase-2 and 5-lipoxygenase inhibitors.”

A number of the above-identified COX-2 inhibitors are prodrugs ofselective COX-2 inhibitors, and exert their action by conversion in vivoto the active and selective COX-2 inhibitors. The active and selectiveCOX-2 inhibitors formed from the above-identified COX-2 inhibitorprodrugs are described in detail in WO 95/00501, published Jan. 5, 1995,WO 95/18799, published Jul. 13, 1995 and U.S. Pat. No. 5,474,995, issuedDec. 12, 1995. Given the teachings of U.S. Pat. No. 5,543,297, entitled:“Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity,” a person of ordinary skill in the art would be able todetermine whether an agent is a selective COX-2 inhibitor or a precursorof a COX-2 inhibitor, and therefore part of the present invention.

An “angiotensin system inhibitor” is an agent that interferes with thefunction, synthesis or catabolism of angiotensin II. These agentsinclude, but are not limited to, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II antagonists, angiotensin II receptorantagonists, agents that activate the catabolism of angiotensin II, andagents that prevent the synthesis of angiotensin I from whichangiotensin II is ultimately derived. The renin-angiotensin system isinvolved in the regulation of hemodynamics and water and electrolytebalance. Factors that lower blood volume, renal perfusion pressure, orthe concentration of Na⁺ in plasma tend to activate the system, whilefactors that increase these parameters tend to suppress its function.

Angiotensin I and angiotensin II are synthesized by the enzymaticrenin-angiotensin pathway. The synthetic process is initiated when theenzyme renin acts on angiotensinogen, pseudoglobulin in blood plasma, toproduce the decapeptide angiotensin I. Angiotensin I is converted byangiotensin converting enzyme (ACE) to angiotensin II (angiotensin-[1-8]octapeptide). The latter is an active pressor substance which has beenimplicated as a causative agent in several forms of hypertension invarious mammalian species, e.g., humans.

Angiotensin (renin-angiotensin) system inhibitors are compounds that actto interfere with the production of angiotensin II from angiotensinogenor angiotensin I or interfere with the activity of angiotensin II. Suchinhibitors are well known to those of ordinary skill in the art andinclude compounds that act to inhibit the enzymes involved in theultimate production of angiotensin II, including renin and ACE. Theyalso include compounds that interfere with the activity of angiotensinII, once produced. Examples of classes of such compounds includeantibodies (e.g., to renin), amino acids and analogs thereof (includingthose conjugated to larger molecules), peptides (including peptideanalogs of angiotensin and angiotensin I), pro-renin related analogs,etc. Among the most potent and useful renin-angiotensin systeminhibitors are renin inhibitors, ACE inhibitors, and angiotensin IIantagonists. In a preferred embodiment of the invention, therenin-angiotensin system inhibitors are renin inhibitors, ACEinhibitors, and angiotensin II antagonists.

“Angiotensin II antagonists” are compounds which interfere with theactivity of angiotensin II by binding to angiotensin II receptors andinterfering with its activity. Angiotensin II antagonists are well knownand include peptide compounds and non-peptide compounds. Mostangiotensin II antagonists are slightly modified congeners in whichagonist activity is attenuated by replacement of phenylalanine inposition 8 with some other amino acid; stability can be enhanced byother replacements that slow degeneration in vivo. Examples ofangiotensin II antagonists include: peptidic compounds (e.g., saralasin,[(San¹⁾(Val⁵)(Ala⁸)] angiotensin-(1-8) octapeptide and related analogs);N-substituted imidazole-2-one (U.S. Pat. No. 5,087,634); imidazoleacetate derivatives including 2-N-butyl-4-chloro-1-(2-chlorobenzile)imidazole-5-acetic acid (see Long et al., J. Pharmacol. Exp. Ther.247(1), 1-7 (1988)); 4, 5, 6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid and analogderivatives (U.S. Pat. No. 4,816,463); N2-tetrazole beta-glucuronideanalogs (U.S. Pat. No. 5,085,992); substituted pyrroles, pyrazoles, andtryazoles (U.S. Pat. No. 5,081,127); phenyl and heterocyclic derivativessuch as 1,3-imidazoles (U.S. Pat. No. 5,073,566); imidazo-fused 7-memberring heterocycles (U.S. Pat. No. 5,064,825); peptides (e.g., U.S. Pat.No. 4,772,684); antibodies to angiotensin II (e.g., U.S. Pat. No.4,302,386); and aralkyl imidazole compounds such as biphenyl-methylsubstituted imidazoles (e.g., EP Number 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(-1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl (35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKF 108566 (E-alpha-2-[2-butyl-1-(carboxyphenyl) methyl]1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid,Smith Kline Beecham Pharmaceuticals, PA); Losartan (DUP753/MK954, DuPontMerck Pharmaceutical Company); Remikirin (RO42-5892, F. Hoffman LaRocheAG); A₂ agonists (Marion Merrill Dow) and certain non-peptideheterocycles (G.D.Searle and Company).

“Angiotensin converting enzyme (ACE), is an enzyme which catalyzes theconversion of angiotensin I to angiotensin II. ACE inhibitors includeamino acids and derivatives thereof, peptides, including di and tripeptides and antibodies to ACE which intervene in the renin-angiotensinsystem by inhibiting the activity of ACE thereby reducing or eliminatingthe formation of pressor substance angiotensin II. ACE inhibitors havebeen used medically to treat hypertension, congestive heart failure,myocardial infarction and renal disease. Classes of compounds known tobe useful as ACE inhibitors include acylmercapto and mercaptoalkanoylprolines such as captopril (U.S. Pat. No. 4,105,776) and zofenopril(U.S. Pat. No. 4,316,906), carboxyalkyl dipeptides such as enalapril(U.S. Pat. No. 4,374,829), lisinopril (U.S. Pat. No. 4,374,829),quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S. Pat. No. 4,587,258),and perindopril (U.S. Pat. No. 4,508,729), carboxyalkyl dipeptide mimicssuch as cilazapril (U.S. Pat. No. 4,512,924) and benazapril (U.S. Pat.No. 4,410,520), phosphinylalkanoyl prolines such as fosinopril (U.S.Pat. No. 4,337,201) and trandolopril.

“Renin inhibitors” are compounds which interfere with the activity ofrenin. Renin inhibitors include amino acids and derivatives thereof,peptides and derivatives thereof, and antibodies to renin. Examples ofrenin inhibitors that are the subject of United States patents are asfollows: urea derivatives of peptides (U.S. Pat. No. 5,116,835); aminoacids connected by nonpeptide bonds (U.S. Pat. No. 5,114,937); di andtri peptide derivatives (U.S. Pat. No. 5,106,835); amino acids andderivatives thereof (U.S. Pat. Nos. 5,104,869 and 5,095,119); diolsulfonamides and sulfinyls (U.S. Pat. No. 5,098,924); modified peptides(U.S. Pat. No. 5,095,006); peptidyl beta-aminoacyl aminodiol carbamates(U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S. Pat. No. 5,075,451);fluorine and chlorine statine or statone containing peptides (U.S. Pat.No. 5,066,643); peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and4,845,079); N-morpholino derivatives (U.S. Pat. No. 5,055,466);pepstatin derivatives (U.S. Pat. No. 4,980,283); N-heterocyclic alcohols(U.S. Pat. No. 4,885,292); monoclonal antibodies to renin (U.S. Pat. No.4,780,401); and a variety of other peptides and analogs thereof (U.S.Pat. Nos. 5,071,837, 5,064,965, 5,063,207, 5,036,054, 5,036,053,5,034,512, and 4,894,437).

Agents that bind to cellular adhesion molecules and inhibit the abilityof white blood cells to attach to such molecules include polypeptideagents. Such polypeptides include polyclonal and monoclonal antibodies,prepared according to conventional methodology. Such antibodies alreadyare known in the art and include anti-ICAM 1 antibodies as well as othersuch antibodies. Significantly, as is well-known in the art, only asmall portion of an antibody molecule, the paratrope, is involved in thebinding of the antibody to its epitope (see, in general, Clark, W. R.(1986) The Experimental Foundations of Modern Immunology, Wiley & Sons,Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed.,Blackwell Scientific Publications, Oxford). The pFc′ and Fc regions, forexample, are effectors of the complement cascade but are not involved inantigen binding. An antibody from which the pFc′ region has beenenzymatically cleaved, or which has been produced without the pFc′region, designated an F(ab′)₂ fragment, retains both of the antigenbinding sites of an intact antibody. Similarly, an antibody from whichthe Fc region has been enzymatically cleaved, or which has been producedwithout the Fc region, designated an Fab fragment, retains one of theantigen binding sites of an intact antibody molecule. Proceedingfurther, Fab fragments consist of a covalently bound antibody lightchain and a portion of the antibody heavy chain denoted Fd. The Fdfragments are the major determinant of antibody specificity (a single FdFragment may be associated with up to ten different light chains withoutaltering antibody specificity) and Fd fragments retain epitope-bindingability in isolation.

Within the antigen-binding portion of an antibody, as is well-know inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(Frs), which maintain the tertiary structure of the paratope (see, ingeneral, Clar, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. Thus, for example, PCT International PublicationNumber WO 92/04381 teaches the production and use of humanized murineRSV antibodies in which at least a portion of the murine FR regions havebeen replaced by FR regions of human origin. Such antibodies, includingfragments of intact antibodies with antigen-binding ability, are oftenreferred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or Fr and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by homologous humanor non-human sequences; chimeric F(ab′)₂ fragment antibodies in whichthe FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have beenreplaced by homologous human or non-human sequences; chimeric Fabfragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or lightchain CDR3 regions have been replaced by homologous human or non-humansequences; and chimeric Fd fragment antibodies in which the FR and/orCDR1 and/or CDR2 regions have been replaced by homologous human ornonhuman sequences. The present invention also includes so-called singlechain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to cellular adhesion molecules. These polypeptides maybe derived also from sources other than antibody technology. Forexample, such polypeptide binding agents can be provided by degeneratepeptide libraries which can be readily prepared in solution, inimmobilized form or as phage display libraries. Combinatorial librariesalso can be synthesized of peptides containing one or more amino acids.Libraries further can be synthesized of peptoids and non-peptidesynthetic moieties.

Phage display can be particularly effective in identifying bindingpeptides useful according to the invention. Briefly, one prepares aphage library (using e.g. m13, fd, or lambda phage), displaying insertsfrom 4 to about 80 amino acid residues using conventional procedures.The inserts may represent, for example, a completely degenerate orbiased array. One then can select phage-bearing inserts which bind tothe cellular adhesion molecule. This process can be repeated throughseveral cycles of reselection of phage that bind to the cellularadhesion molecule. Repeated rounds lead to enrichment of phage bearingparticular sequences. DNA sequences analysis can be conducted toidentify the sequences of the expressed polypeptides. The minimal linearportion of the sequence that binds to the cellular adhesion molecule canbe determined. One can repeat the procedure using a biased librarycontaining inserts containing part of all of the minimal linear portionplus one or more additional degenerate residues upstream or downstreamthereof. Yeast two-hybrid screening methods also may be used to identifypolypeptides that bind to the cellular adhesion molecules. Thus,cellular adhesion molecules, or a fragment thereof, can be used toscreen peptide libraries, including phage display libraries, to identifyand select peptide binding partners of the cellular adhesion molecules.

An “infection” or “infectious disease” as used herein, refers to adisorder arising from the invasion of a host, superficially, locally, orsystemically, by an infectious microorganism. Infectious microorganismsinclude bacteria, viruses, parasites and fungi. The term “sepsis” refersto the clinical condition in which infective agents (bacteria,pathogenic fungi) or products of infection (bacterial toxins) enter theblood circulation and profoundly affects the patient's blood pressure,heart rate, and body temperature.

Bacteria are unicellular organisms which multiply asexually by binaryfission. They are classified and named based on their morphology,staining reactions, nutrition and metabolic requirements, antigenicstructure, chemical composition, and genetic homology. Bacteria can beclassified into three groups based on their morphological forms,spherical (coccus), straight-rod (bacillus) and curved or spiral rod(vibrio, campylobacter, spirillum, and spirochaete). Bacteria are alsomore commonly characterized based on their staining reactions into twoclasses of organisms, gram-positive and gram-negative. Gram refers tothe method of staining which is commonly performed in microbiology labs.Gram-positive organisms retain the stain following the stainingprocedure and appear a deep violet color. Gram-negative organisms do notretain the stain but take up the counter-stain and thus appear pink.

Bacteria have two main structural components, a rigid cell wall andprotoplast (material enclosed by the cell wall). The protoplast includescytoplasm and genetic material. Surrounding the protoplast is thecytoplasmic membrane which includes some of the cell respiratory enzymesand is responsible for the permeability of bacteria and transport ofmany small molecular weight substances. The cell wall surrounding thecytoplasmic membrane and protoplast is composed of mucopeptides whichinclude complex polymers of sugars cross-linked by peptide chains ofamino acids. The wall is also composed of polysaccharides and teichoicacids.

Infectious bacteria include, but are not limited to, gram negative andgram positive bacteria. Gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria species (e.g. M. tuberculosis, M. avium, M. intracellulare,M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic species), Streptococcuspneumoniae, pathogenic Campylobacter species, Enterococcus species,Haemophilus influenzae, Bacillus antracis, Corynebacterium diphtheriae,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides species, Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospiraspecies, Rickettsia species, and Actinomyces israelli. Additionalexemplary bacteria are Mycoplasma, e.g. Mycoplasma pneumoniae,Chlamydophila, e.g. Chlamydophila pneumoniae, Bartonella species, andTropheryma whippelii.

Viruses are small infectious agents which contain a nucleic acid coreand a protein coat, but are not independently living organisms. A viruscannot survive in the absence of a living cell within which it canreplicate. Viruses enter specific living cells either by endocytosis ordirect injection of DNA (phage) and multiply, causing disease. Themultiplied virus can then be released and infect additional cells. Someviruses are DNA-containing viruses and other are RNA-containing viruses.

Once the virus enters the cell it can cause a variety of physiologicaleffects. One effect is cell degeneration, in which the accumulation ofvirus within the cell causes the cell to die and break into pieces andrelease the virus. Another effect is cell fusion, in which infectedcells fuse with neighboring cells to produce syncytia. Other types ofvirus cause cell proliferation which results in tumor formation.

Specific examples of viruses that have been found in humans include butare not limited to: Retroviridae (e.g. human immunodeficiency viruses,such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, orHIV-III); and other isolates, such as HIV-LP); Picornaviridae (e.g.polio viruses, hepatitis A virus; enteroviruses, human Coxsackieviruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains thatcause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses,rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses,yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae(e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g.ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumpsvirus, measles virus, respiratory syncytial virus); Orthomyxoviridae(e.g. influenza viruses); Bunyaviridae (e.g. Hantaan viruses,bunyaviruses, phleboviruses and Nairo viruses); Arenaviridae(hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviursesand rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus);Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus(CMV), herpes virus); Poxyiridae (variola viruses, vaccinia viruses, poxviruses); and Iridoviridae (e.g. African swine fever virus); andunclassified viruses (e.g., the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

In addition to viruses that infect human subjects causing humandisorders, the invention is also useful for treating other non-humanvertebrates. Non-human vertebrates are also capable of developinginfections which can be prevented or treated with the combinations ofaziridino compounds and anti-microbials disclosed herein. For instance,in addition to the treatment of infectious human diseases, the methodsof the invention are useful for treating or preventing infections ofnon-human animals.

Infectious virus of both human and non-human vertebrates, includeretroviruses, RNA viruses and DNA viruses. This group of retrovirusesincludes both simple retroviruses and complex retroviruses. The simpleretroviruses include the subgroups of B-type retroviruses, C-typeretroviruses and D-type retroviruses. An example of a B-type retrovirusis mouse mammary tumor virus (MMTV). The C-type retroviruses includesubgroups C-type group A (including Rous sarcoma virus (RSV), avianleukemia virus (ALV), and avian myeloblastosis virus (AMV)) and C-typegroup B (including murine leukemia virus (MLV), feline leukemia virus(FeLV), murine sarcoma virus (MSV), gibbon ape leukemia virus (GALV),spleen necrosis virus (SNV), reticuloendotheliosis virus (RV) and simiansarcoma virus (SSV)). The D-type retroviruses include Mason-Pfizermonkey virus (MPMV) and simian retrovirus type 1 (SRV-1). The complexretroviruses include the subgroups of lentiviruses, T-cell leukemiaviruses and the foamy viruses. Lentiviruses include HIV-1, but alsoinclude HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV),and equine infectious anemia virus (EIAV). The T-cell leukemia virusesinclude HTLV-1, HTLV-II, simian T-cell leukemia virus (STLV), and bovineleukemia virus (BLV). The foamy viruses include human foamy virus (HFV),simian foamy virus (SFV) and bovine foamy virus (BFV).

Examples of other RNA viruses that are pathogens in vertebrate animalsinclude, but are not limited to, the following: members of the familyReoviridae, including the genus Orthoreovirus (multiple serotypes ofboth mammalian and avian retroviruses), the genus Orbivirus (Bluetonguevirus, Eugenangee virus, Kemerovo virus, African horse sickness virus,and Colorado Tick Fever virus), the genus Rotavirus (human rotavirus,Nebraska calf diarrhea virus, murine rotavirus, simian rotavirus, bovineor ovine rotavirus, avian rotavirus); the family Picornaviridae,including the genus Enterovirus (poliovirus, Coxsackie virus A and B,enteric cytopathic human orphan (ECHO) viruses, hepatitis A virus,Simian enteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirusmuris, Bovine enteroviruses, Porcine enteroviruses, the genusCardiovirus (Encephalomyocarditis virus (EMC), Mengovirus), the genusRhinovirus (Human rhinoviruses including at least 113 subtypes; otherrhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); thefamily Calciviridae, including Vesicular exanthema of swine virus, SanMiguel sea lion virus, Feline picornavirus and Norwalk virus; the familyTogaviridae, including the genus Alphavirus (Eastern equine encephalitisvirus, Semliki forest virus, Sindbis virus, Chikungunya virus,O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus), the genus Flavirus (Mosquitoborne yellow fever virus, Dengue virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley encephalitis virus, West Nilevirus, Kunjin virus, Central European tick borne virus, Far Eastern tickborne virus, Kyasanur forest virus, Louping III virus, Powassan virus,Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), thegenus Pestivirus (Mucosal disease virus, Hog cholera virus, Borderdisease virus); the family Bunyaviridae, including the genus Bunyavirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusinfluenza virus (influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family Paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus of mice); the family Rhabdoviridae,including the genus Vesiculovirus (VSV), Chandipura virus, Flanders-HartPark virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses,and two probable Rhabdoviruses (Marburg virus and Ebola virus); thefamily Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus,Human enteric corona virus, and Feline infectious peritonitis (Felinecoronavirus).

Illustrative DNA viruses that infect vertebrate animals include, but arenot limited to: the family Poxyiridae, including the genus Orthopoxvirus(Variola major, Variola minor, Monkeypox, Vaccinia, Cowpox, Buffalopox,Rabbitpox, Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), thegenus Avipoxvirus (Fowlpox, other avian poxvirus), the genusCapripoxvirus (sheeppox, goatpox), the genus Suipoxvirus (Swinepox), thegenus Parapoxvirus (contagious postular dermatitis virus, pseudocowpox,bovine papular stomatitis virus); the family Iridoviridae (African swinefever virus, Frog viruses 2 and 3, Lymphocystis virus of fish); thefamily Herpesviridae, including the alpha-Herpesviruses (Herpes SimplexTypes 1 and 2, Varicella-Zoster, Equine abortion virus, Equine herpesvirus 2 and 3, pseudorabies virus, infectious bovinekeratoconjunctivitis virus, infectious bovine rhinotracheitis virus,feline rhinotracheitis virus, infectious laryngotracheitis virus) theBeta-herpesviruses (Human cytomegalovirus and cytomegaloviruses ofswine, monkeys and rodents); the gamma-herpesviruses (Epstein-Barr virus(EBV), Marek's disease virus, Herpes saimiri, Herpesvirus ateles,Herpesvirus sylvilagus, guinea pig herpes virus, Lucke tumor virus); thefamily Adenoviridae, including the genus Mastadenovirus (Human subgroupsA,B,C,D,E and ungrouped); simian adenoviruses (at least 23 serotypes),infectious canine hepatitis, and adenoviruses of cattle, pigs, sheep,frogs and many other species, the genus Aviadenovirus (Avianadenoviruses); and non-cultivatable adenoviruses; the familyPapoviridae, including the genus Papillomavirus (Human papillomaviruses, bovine papilloma viruses, Shope rabbit papilloma virus, andvarious pathogenic papilloma viruses of other species), the genusPolyomavirus (polyomavirus, Simian vacuolating agent (SV-40), Rabbitvacuolating agent (RKV), K virus, BK virus, JC virus, and other primatepolyoma viruses such as Lymphotrophic papilloma virus); the familyParvoviridae including the genus Adeno-associated viruses, and the genusParvovirus (Feline panleukopenia virus, bovine parvovirus, canineparvovirus, Aleutian mink disease virus, etc).

Parasites are organisms which depend upon other organisms in order tosurvive and thus must enter, or infect, another organism to continuetheir life cycle. The infected organism, i.e., the host, provides bothnutrition and habitat to the parasite. The term “parasite” as usedherein refers to protozoa, helminths, and ectoparasitic arthropods(e.g., ticks, mites, etc.). Protozoa are single celled organisms whichcan replicate both intracellularly and extracellularly, particularly inthe blood, intestinal tract or the extracellular matrix of tissues.Helminths are multicellular organisms which almost always areextracellular (the exception being Trichinella). Helminths normallyrequire exit from a primary host and transmission into a secondary hostin order to replicate. In contrast to these aforementioned classes,ectoparasitic arthropods form a parasitic relationship with the externalsurface of the host body.

Parasites can be classified based on whether they are intracellular orextracellular. An “intracellular parasite” as used herein is a parasitewhose entire life cycle is intracellular. Examples of humanintracellular parasites include Leishmania, Plasmodium, Trypanosomacruzi, Toxoplasma gondii, Babesia, and Trichinella spiralis. An“extracellular parasite” as used herein is a parasite whose entire lifecycle is extracellular. Extracellular parasites capable of infectinghumans include Entamoeba histolytica, Giardia lamblia, Enterocytozoonbieneusi, Naegleria and Acanthamoeba as well as most helminths. Yetanother class of parasites is defined as being mainly extracellular butwith an obligate intracellular existence at a critical stage in theirlife cycles. Such parasites are referred to herein as “obligateintracellular parasites”. These parasites may exist most of their livesor only a small portion of their lives in an extracellular environment,but they all have at lest one obligate intracellular stage in their lifecycles. This latter category of parasites includes Trypanosomarhodesiense and Trypanosoma gambiense, Isospora, Cryptosporidium,Eimeria, Neospora, Sarcocystis, and Schistosoma. In one aspect, theinvention relates to the prevention and treatment of infection resultingfrom intracellular parasites and obligate intracellular parasites whichhave at least in one stage of their life cycle that is intracellular. Insome embodiments, the invention is directed to the prevention ofinfection from obligate intracellular parasites which are predominantlyintracellular. An exemplary and non-limiting list of parasites for someaspects of the invention is provided herein.

Blood-borne and/or tissues parasites include Plasmodium, Babesiamicroti, Babesia divergens, Leishmania tropica, Leishmania, Leishmaniabraziliensis, Leishmania donovani, Trypanosoma gambiense and Trypanosomarhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas'disease), and Toxoplasma gondii. Typical parasites infecting horses areGasterophilus; Eimeria leuckarti, Giardia; Tritrichomonas equi; Babesia(RBCs), Theileria equi; Trypanosoma; Klossiella equi; Sarcocystis.

Typical parasites infecting swine include Eimeria bebliecki, Eimeriascabra, Isospora suis, Giardia; Balantidium coli, Entamoeba histolytica;Toxoplasma gondii and Sarcocystis, and Trichinella spiralis.

The major parasites of dairy and beef cattle include Eimeria,Cryptosporidium, Giardia; Toxoplasma gondii; Babesia bovis (RBCs),Babesia bigemina (RBCs), Trypanosoma (plasma), Theileria (RBC);Theileria parva (lymphocytes); Tritrichomonas foetus; and Sarcocystis.

Typical parasites infecting sheep and goats include Eimeria,Cryptosporidium, Giardia; Toxoplasma gondii; Babesia (RBC), Trypanosoma(plasma), Theileria (RBC); and Sarcocystis.

Typical parasitic infections in poultry include coccidiosis caused byEimeria acervulina, E. necatrix, E. tenella, Isospora and Eimeriatruncata; histomoniasis, caused by Histomonas meleagridis and Histomonasgallinarum; trichomoniasis caused by Trichomonas gallinae; andhexamitiasis caused by Hexamita meleagridis. Poultry can also beinfected Emeria maxima, Emeria meleagridis, Eimeria adenoeides, Eimeriameleagrimitis, Cryptosporidium, Eimeria brunetti, Emeria adenoeides,Leucocytozoon, Plasmodium, Hemoproteus meleagridis, Toxoplasma gondiiand Sarcocystis.

Parasitic infections also pose serious problems in laboratory researchsettings involving animal colonies. Some examples of laboratory animalsintended to be treated, or in which parasite infection is sought to beprevented, by the methods of the invention include mice, rats, rabbits,guinea pigs, nonhuman primates, as well as the aforementioned swine andsheep.

Typical parasites in mice include Leishmania, Plasmodium berghei,Plasmodium yoelii, Giardia muris, Hexamita muris; Toxoplasma gondii;Trypanosoma duttoni (plasma); Klossiella muris; Sarcocystis. Typicalparasites in rats include Giardia muris, Hexamita muris; Toxoplasmagondii; Trypanosoma lewisi (plasma); Trichinella spiralis; andSarcocystis. Typical parasites in rabbits include Eimeria; Toxoplasmagondii; Nosema cuniculi; Eimeria stiedae, and Sarcocystis. Typicalparasites of the hamster include Trichomonas; Toxoplasma gondii;Trichinella spiralis; and Sarcocystis. Typical parasites in the guineapig include Balantidium caviae; Toxoplasma gondii; Klossiella caviae;and Sarcocystis.

Fungi are eukaryotic organisms, only a few of which cause infection invertebrate mammals. Because fungi are eukaryotic organisms, they differsignificantly from prokaryotic bacteria in size, structuralorganization, life cycle and mechanism of multiplication. Fungi areclassified generally based on morphological features, modes ofreproduction and culture characteristics. Although fungi can causedifferent types of disease in subjects, such as respiratory allergiesfollowing inhalation of fungal antigens, fungal intoxication due toingestion of toxic substances, such as amatatoxin and phallotoxinproduced by poisonous mushrooms and aflotoxins, produced by aspergillusspecies, not all fungi cause infectious disease.

Infectious fungi can cause systemic or superficial infections. Primarysystemic infection can occur in normal healthy subjects andopportunistic infections, are most frequently found inimmuno-compromised subjects. The most common fungal agents causingprimary systemic infection include Blastomyces, Coccidioides, andHistoplasma. Common fungi causing opportunistic infection inimmuno-compromised or immunosuppressed subjects include, but are notlimited to, Candida albicans (an organism which is normally part of therespiratory tract flora), Cryptococcus neoformans (sometimes in normalflora of respiratory tract), and various Aspergillus species. Systemicfungal infections are invasive infections of the internal organs. Theorganism usually enters the body through the lungs, gastrointestinaltract, or intravenous lines. These types of infections can be caused byprimary pathogenic fungi or opportunistic fungi.

Superficial fungal infections involve growth of fungi on an externalsurface without invasion of internal tissues. Typical superficial fungalinfections include cutaneous fungal infections involving skin, hair, ornails. An example of a cutaneous infection is Tinea infections, such asringworm, caused by Dermatophytes, such as Microsporum or Traicophytonspecies, i.e., Microsporum canis, Microsporum gypsum, Tricofitin rubrum.Examples of fungi include: Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans.

Diseases associated with fungal infection include aspergillosis,blastomycosis, camdidiais, chromoblastomycosis, coccidioidomycosis,cryptococcosis, fungal eye infections, fungal hair, nail, and skininfections, histoplasmosis, lobomycosis, mycetoma, otomycosis,paracoccidioidomycosis, penicilliosis, marneffeii, phaeohyphomycosis,rhinosporidioisis, sporotrichosis, and zygomycosis.

Aspergillosis is a disease caused by the fungi of the genus Aspergillus,which can lead to mild or severe disease, generally depending on factorssuch as the status of the host immune system. Aspergillus frequentlyarises as an opportunistic infection in patients havingimmune-suppressive diseases, or being treated with chemotherapy. Someforms of aspergillus can be treated with prednisone, disodiumchromoglycat, nystatin, amphotericin B, itraconazole, or voriconazole.

Blastomycosis is a fungal infection arising from the organismBlastomyces dermatitis. The infection initiates in the lungs and usuallyis disseminated to other body sites, especially the skin and bone. It istreated by amphotericin B, hydroxystilbamidine, itraconazole andvoriconazole. When amphotericin B is used, at least 1.5 grams must begiven to avoid relapse. However, when the drug is administered incombination with the aziridino compounds, lower doses can be givenwithout a relapse. Generally hydroxystilbamidine has been used fortreating the cutaneous form of the disease but not other forms. Whencombined with aziridino compounds in the combination compositions of theinvention, it can also be used for the treatment of other forms, as wellas in lower doses for the cutaneous form.

Candidiasis is a fungal infection caused by a member of the genusCandida. The disease can be in the form of allergic, cutaneous,mucocutaneous, or systemic candidiasis. Nystatin is used for thetreatment of the cutaneous, mucocutaneous, and allergic diseases.Amphotericin B is useful for treating this systemic disease. Other drugsuseful for the treatment include 5-fluorocytosine, fluconazole,itraconazole and voriconazole.

Chromoblastomycosis is a chronic infection of the skin and subcutaneoustissue. Although the infection is usually localized, parts candisseminate systemically and in particular to the brain. Itraconazoleand terbinafine are the drugs used to treat this infection. Theprincipal fungi causing this infection are Cladophialophora, Carrionii,Fonsecaea, Compacta, Fonsecaeapedrosoi, Phialophora, Verruceosa,Rhinocladiella, and Aquasbera.

Coccidioidomycosis is a fungal disease of the respiratory tract whichcan be acute, chronic, severe or fatal. The disease is primarily causedby Coccidioides immitis. Amphotericin B, itraconazole, fluconazole,ketaconazole, and voriconazole are anti-fungal agents that are used forthe treatment of this disorder.

Cryptococcosis is a fungal disorder caused by Cryptococcus norformans orFilobasidiella neoformans. The disease can take the form of a chronic,subacute, acute, pulmonary, systemic, or meningitic disease, followingprimary infection in the lungs. If the disease spreads from the lungs tothe central nervous system, it is usually treated immediately withamphotericin B and/or 5-fluorocytosine and in some cases fluconazole.

Fungal infections of the eye include mycotic keratitis, and endogenousor extension oculomycosis. Mycotic keratitis is caused by a variety offungi including Acremonium, Aspergillus, Bipolaris, Candida albicans,Curvularia, Exserohilum, Fusarium, and Lasiodiplodia. Amphotericin B isnot used for treatment because it irritates the infected tissue. Drugsuseful for treating mycotic keratitis include pimaricin and fluconazole.Oculomycosis is generally caused by Candida albicans or rhizopus,arrhizus. Amphotericin B is the anti-fungal agent used for treatment.

Fungal infections of the hair, nail, and skin include onychomycosis,piedra, pityriasis versicolor, tinea barbae, tinea capitis, tineacorporis, tinea cruris, tinea faosa, tinea nigra, tinea unguium.Onychomycosis, which is generally caused by fungi such as Acremonium,Aspergillus, Candida, Fusarium, Scopulariopisis, Onychocola, andScytalidium, can be treated with itraconazole, turbinifine, amphotericinB, gentian violet, resorcin, iodine, nystatin, thiabendazole, andglutarardehyde. Piedra, which is a colonization of the hair shaft tobifungal organisms such as Piedraia and Trichosporin, can be treatedwith keratolytic agents, mild fungicides, fluconazole, and itraconazole.The tineas are various forms of ringworm colonizing different bodilyregions. These diseases are generally caused by fungi such asMicrosporum, Trichophyton, and Epidermophyton. The tineas can be treatedwith keratolytic agents, intraconazole, turbinifine, tolnaftate,clotrimazole, miconazole, econazole, and ketaconzole.

Histoplasmosis (capsulati and duboisii) are fungal infections caused byHistoplasma and Ajellomyces. Histoplasmosis capsulati can adequately betreated with amphotericin B, itraconazole or voriconazole. If thesubject being treated has AIDS, fluconazole is usually used.Histoplasmosis duboisii once it becomes disseminated, especially to theliver or spleen, is very difficult to treat. Amphotericin B,itraconazole, fluconazole, and voriconazole are used. When thesecompounds are combined with the aziridino compounds of the invention,prognosis is improved.

Lobomycosis is a fungal infection caused by Lacazia loboi. Lobomycosisis a cutaneous infection which develops into lesions which can beremoved by surgery. There are not drugs specifically used for thisdisorder. Mycetoma is an infection caused by a variety of fungiincluding Eumycotic, Acromonium, Aspergillus, Exophiala, Leptos Phaeria,Madurella, Neotestudina, Pseudallesheria, and Pyrenochieta. The diseaseinvolves lesions of the cutaneous and subcutaneous tissues, which canrupture and spread to surrounding tissues. The mycetomas can be treatedwith ketoconazole, in combination with surgery.

Otomycosis is a fungal ear infection caused by Aspergillus or candida.The infection is a superficial infection of the outer ear canal, whichis characterized by inflammation, pruritus, scaling, and severdiscomfort. It is a chronic recurring mycosis.

Paracoccidioidomycosis is a fungal infection cause by Paracoccidioidesbrasiliensis. The disease originates as a pulmonary infection and candisseminate into the nasal, buccal, and gastrointestinal mucosa.Amphotericin B and sulfonamides are generally used to treat the disease.

Phaeohyphomycosis is a fungal infection caused by a variety of fungiincluding Cladophialophora, Curvularia, Bipolaris, Exserohilum,Exophiala, Scedosporium, Ochroconis, Coniothyrium, Phialophora,Wangiella, and Lasiodiplodia. The infection can be localized or caninvade various tissues including the brain, bone, eyes, and skin.Invasion of the brain or bone can be lethal. Generally,phaeohyphomycosis is treated with amphotericin B and phyfluorocytozineor intaconazole. Rhinosporidiosis is an infection of the mucus membranecaused by Rhinosporidium seeberi. Local injection of amphotericin B isused as treatment.

Sporotrichosis is a chronic infection of the cutaneous tissues,subcutaneous tissues, or lymph system. The infection may also spread totissues such as bone, muscle, CNS, lungs, and/or genitourinary system.Usually the fungi Sporothrix schenckii is inhaled or passed through alesion in the skin. Sporotrichosis is usually treated with oralpotassium iodide, amphotericin B, or 5-fluorocytozine.

Zygomycosis is a chronic infection caused by Conidobolus andBasidiobolus ranarum. The disease is treated by potassium iodide and/oramphotericin B.

Other medically relevant microorganisms and the diseases they cause havebeen described extensively in the literature, e.g., see C. G. A. Thomas,Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entirecontents of which is hereby incorporated by reference. Each of theforegoing lists is illustrative, and is not intended to be limiting.

The methods of the invention involve, in some aspects, combinations ofcompounds that are inhibitors of cellular necrosis (e.g., heterocyclicthiohydantoin, hydantoin, oxazolidinone, thioxo-oxazolidinone,pyrimidinone, or oxazinanone compounds, or combinations thereof) withanti-microbial agents for the treatment or prevention of infectiousdisease. An “anti-microbial agent”, as used herein, refers to anaturally-occurring or synthetic compound which is capable of killing orinhibiting infectious microorganisms. The type of anti-microbial agentuseful according to the invention will depend upon the type ofmicroorganism with which the subject is infected or at risk of becominginfected. It is contemplated that several different kinds ofanti-microbial agents can be combined with the aziridino compounds tomake compositions useful for treating multifactorial diseases (e.g., HIVinfection with opportunistic fungal infections).

One type of anti-microbial agent is an antibacterial agent.Antibacterial agents kill or inhibit the growth or function of bacteria.A large class of antibacterial agents is antibiotics. Antibiotics, whichare effective for killing or inhibiting a wide range of bacteria, arereferred to as broad spectrum antibiotics. Other types of antibioticsare predominantly effective against the bacteria of the classgram-positive or gram-negative. These types of antibiotics are referredto as narrow spectrum antibiotics. Other antibiotics which are effectiveagainst a single organism or disease and not against other types ofbacteria, are referred to as limited spectrum antibiotics.

Antibacterial agents are sometimes classified based on their primarymode of action. In general, antibacterial agents are cell wall synthesisinhibitors, cell membrane inhibitors, protein synthesis inhibitors,nucleic acid synthesis or functional inhibitors, and competitiveinhibitors. Cell wall synthesis inhibitors inhibit a step in the processof cell wall synthesis, and in general in the synthesis of bacterialpeptidoglycan. Cell wall synthesis inhibitors include β-lactamantibiotics, natural penicillins, semi-synthetic penicillins,ampicillin, clavulanic acid, cephalolsporins, and bacitracin.

The β-lactams are antibiotics containing a four-membered β-lactam ringwhich inhibits the last step of peptidoglycan synthesis. β-lactamantibiotics can be synthesized or natural. The natural antibiotics aregenerally produced by two groups of fungi, Penicillium andCephalosporium molds. The β-lactam antibiotics produced by Penicilliumare the natural penicillins, such as penicillin G or penicillin V. Theseare produced by fermentation of Penicillium chrysogenum. The naturalpenicillins have a narrow spectrum of activity and are generallyeffective against Streptococcus, Gonococcus, and Staphylococcus. Othertypes of natural penicillins, which are also effective againstgram-positive bacteria, include penicillins F, X, K, and O.

Semi-synthetic penicillins are generally modifications of the molecule6-aminopenicillanic acid produced by a mold. The 6-aminopenicillanicacid can be modified by addition of side chains which producepenicillins having broader spectrums of activity than naturalpenicillins or various other advantageous properties. Some types ofsemi-synthetic penicillins have broad spectrums against gram-positiveand gram-negative bacteria, but are inactivated by penicillinase. Thesesemi-synthetic penicillins include ampicillin, carbenicillin, oxacillin,azlocillin, mezlocillin, and piperacillin. Other types of semi-syntheticpenicillins have narrower activities against gram-positive bacteria, buthave developed properties such that they are not inactivated bypenicillinase. These include, for instance, methicillin, dicloxacillin,and nafcillin. Some of the broad spectrum semi-synthetic penicillins canbe used in combination with β-lactamase inhibitors, such as clavulanicacids and sulbactam. The β-lactamase inhibitors do not haveanti-microbial action but they function to inhibit penicillinase, thusprotecting the semi-synthetic penicillin from degradation.

Another type of β-lactam antibiotic is the cephalosporins.Cephalosporins are produced by Cephalosporium molds, and have a similarmode of action to penicillin. They are sensitive to degradation bybacterial β-lactamases, and thus, are not always effective alone.Cephaloisporins, however, are resistant to penicillinase. They areeffective against a variety of gram-positive and gram-negative bacteria.Cephalolsporins include, but are not limited to, cephalothin,cephapirin, cephalexin, cefamandole, cefaclor, cefazolin, cefuroxine,cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone,cefoperazone, ceftazidine, and moxalactam.

Bacitracin is another class of antibiotics which inhibit cell wallsynthesis. These antibiotics, produced by Bacillus species, prevent cellwall growth by inhibiting the release of muropeptide subunits orpeptidoglycan from the molecule that delivers the subunit to the outsideof the membrane. Although bacitracin is effective against gram-positivebacteria, its use is limited in general to topical administrationbecause of its high toxicity. Since lower effective doses of bacitracincan be used when the compound is administered with the aziridinocompounds in accordance with the invention, this compound can be usedsystemically and the toxicity reduced.

Carbapenems are another type of broad spectrum β-lactam antibiotic,which is capable of inhibiting cell wall synthesis. Examples ofcarbapenems include, but are not limited to, imipenems. Monobactems arealso broad spectrum β-lactam antibiotics, and include, euztreonam. Anantibiotic produced by Streptomyces, vancomycin, is also effectiveagainst gram-positive bacteria by inhibiting cell membrane synthesis.

Another class of anti-bacterial agents is the anti-bacterial agents thatare cell membrane inhibitors. These compounds disorganize the structureor inhibit the function of bacterial membranes. Alteration of thecytoplasmic membrane of bacteria results in leakage of cellularmaterials from the cell. Compounds that inhibit or interfere with thecell membrane cause death of the cell because the integrity of thecytoplasmic and outer membranes is vital to bacteria. One problem withanti-bacterial agents that are cell membrane inhibitors is that they canproduce effects in eukaryotic cells as well as bacteria because of thesimilarities in phospholipids in bacterial and eukaryotic membranes.Thus these compounds are rarely specific enough to permit thesecompounds to be used systemically and prevent the use of high doses forlocal administration.

One clinically useful anti-bacterial agent that is a cell membraneinhibitor is Polymyxin, produced by Bacillus polymyxis. Polymyxinsinterfere with membrane function by binding to membrane phospholipids.Polymyxin is effective mainly against Gram-negative bacteria and isgenerally used in severe Pseudomonas infections or Pseudomonasinfections that are resistant to less toxic antibiotics. It is also usedin some limited instances topically. The limited use of this agent isdue to the severe side effects associated with systemic administration,such as damage to the kidney and other organs.

Other cell membrane inhibitors include Amphotericin B and Nystatinproduced by the bacterium Streptomyces which are also anti-fungalagents, used predominantly in the treatment of systemic fungalinfections and Candida yeast infections respectively. Imidazoles,produced by the bacterium Streptomyces, are another class of antibioticthat is a cell membrane inhibitor. Imidazoles are used as bacterialagents as well as anti-fungal agents, e.g., used for treatment of yeastinfections, dermatophytic infections, and systemic fungal infections.Imidazoles include but are not limited to clotrimazole, miconazole,ketoconazole, itraconazole, and fluconazole.

Many anti-bacterial agents are protein synthesis inhibitors. Thesecompounds prevent bacteria from synthesizing structural proteins andenzymes and thus cause inhibition of bacterial cell growth or functionor cell death. In general these compounds interfere with the processesof transcription or translation. Anti-bacterial agents that blocktranscription include but are not limited to Rifampins, produced by thebacterium Streptomyces and Ethambutol, a synthetic chemical. Rifampins,which inhibit the enzyme RNA polymerase, have a broad spectrum activityand are effective against gram-positive and gram-negative bacteria aswell as Mycobacterium tuberculosis. Ethambutol is effective againstMycobacterium tuberculosis.

Anti-bacterial agents which block translation interfere with bacterialribosomes to prevent mRNA from being translated into proteins. Ingeneral this class of compounds includes but is not limited totetracyclines, chloramphenicol, the macrolides (e.g. erythromycin) andthe aminoglycosides (e.g. streptomycin).

Some of these compounds bind irreversibly to the 30S ribosomal subunitand cause a misreading of the mRNA, e.g., the aminoglycosides. Theaminoglycosides are a class of antibiotics which are produced by thebacterium Streptomyces, such as, for instance streptomycin, kanamycin,tobramycin, amikacin, and gentamicin. Aminoglycosides have been usedagainst a wide variety of bacterial infections caused by Gram-positiveand Gram-negative bacteria. Streptomycin has been used extensively as aprimary drug in the treatment of tuberculosis. Gentamicin is usedagainst many strains of Gram-positive and Gram-negative bacteria,including Pseudomonas infections, especially in combination withtobramycin. Kanamycin is used against many Gram-positive bacteria,including penicillin-resistant Staphylococci. One side effect ofaminoglycosides that has limited their use clinically is that at dosageswhich are essential for efficacy, prolonged use has been shown to impairkidney function and cause damage to the auditory nerves leading todeafness.

Another type of translation inhibitor anti-bacterial agent is thetetracyclines. The tetracyclines bind reversibly to the 30s ribosomalsubunit and interfere with the binding of charged tRNA to the bacterialribosome. The tetracyclines are a class of antibiotics, produced by thebacterium Streptomyces, that are broad-spectrum and are effectiveagainst a variety of gram-positive and gram-negative bacteria. Examplesof tetracyclines include tetracycline, minocycline, doxycycline, andchlortetracycline. They are important for the treatment of many types ofbacteria but are particularly important in the treatment of Lymedisease.

Anti-bacterial agents such as the macrolides bind reversibly to the 50Sribosomal subunit and inhibits elongation of the protein by peptidyltransferase or prevents the release of uncharged tRNA from the bacterialribosome or both. The macrolides contain large lactone rings linkedthrough glycoside bonds with amino sugars. These compounds includeerythromycin, roxithromycin, clarithromycin, oleandomycin, andazithromycin. Erythromycin is active against most Gram-positivebacteria, Neisseria, Legionella and Haemophilus, but not against theEnterobacteriaceae. Lincomycin and clindamycin, which block peptide bondformation during protein synthesis, are used against gram-positivebacteria.

Another type of translation inhibitor is chloramphenicol.Chloramphenicol binds the 70S ribosome inhibiting the bacterial enzymepeptidyl transferase thereby preventing the growth of the polypeptidechain during protein synthesis. Chloramphenicol can be prepared fromStreptomyces or produced entirely by chemical synthesis. One seriousside effect associated with chloramphenicol is aplastic anemia. Aplasticanemia develops at doses of chloramphenicol which are effective fortreating bacteria in a small proportion (1/50,000) of patients.Chloramphenicol which was once a highly prescribed antibiotic is nowseldom uses as a result of the deaths from anemia. Because of itseffectiveness it is still used in life-threatening situations (e.g.typhoid fever). By combining chloramphenicol with aziridino compounds asdescribed herein, chloramphenicol can again be used as an anti-bacterialagent because the action of the aziridino compounds allows a lower doseof the chloramphenicol to be used, a dose that does not produce sideeffects.

Some anti-bacterial agents disrupt nucleic acid synthesis or function,e.g., bind to DNA or RNA so that their messages cannot be read. Theseinclude but are not limited to quinolones and co-trimoxazole, bothsynthetic chemicals and rifamycins, a natural or semi-syntheticchemical. The quinolones block bacterial DNA replication by inhibitingthe DNA gyrase, the enzyme needed by bacteria to produce their circularDNA. They are broad spectrum and examples include norfloxacin,ciprofloxacin, enoxacin, nalidixic acid and temafloxacin. Nalidixic acidis a bactericidal agent that binds to the DNA gyrase enzyme(topoisomerase) which is essential for DNA replication and allowssupercoils to be relaxed and reformed, inhibiting DNA gyrase activity.The main use of nalidixic acid is in treatment of lower urinary tractinfections (UTI) because it is effective against several types ofGram-negative bacteria such as E. coli, Enterobacter aerogenes, Kpneumoniae and Proteus species which are common causes of UTI.Co-trimoxazole is a combination of sulfamethoxazole and trimethoprim,which blocks the bacterial synthesis of folic acid needed to make DNAnucleotides. Rifampicin is a derivative of rifamycin that is activeagainst Gram-positive bacteria (including Mycobacterium tuberculosis andmeningitis caused by Neisseria meningitidis) and some Gram-negativebacteria. Rifampicin binds to the beta subunit of the polymerase andblocks the addition of the first nucleotide which is necessary toactivate the polymerase, thereby blocking mRNA synthesis.

Another class of anti-bacterial agents is compounds that function ascompetitive inhibitors of bacterial enzymes. The competitive inhibitorsare mostly all structurally similar to a bacterial growth factor andcompete for binding but do not perform the metabolic function in thecell. These compounds include sulfonamides and chemically modified formsof sulfanilamide which have even higher and broader antibacterialactivity. The sulfonamides (e.g. gantrisin and trimethoprim) are usefulfor the treatment of Streptococcus pneumoniae, beta-hemolyticstreptococci and E. coli, and have been used in the treatment ofuncomplicated UTI caused by E. coli, and in the treatment ofmeningococcal meningitis.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleoside analogues), maturation of new virus proteins(e.g. protease inhibitors), and budding and release of the virus.

Nucleotide analogues are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogues are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleotide analoguesinclude, but are not limited to, acyclovir (used for the treatment ofherpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, and zidovudine (azidothymidine).

The interferons are cytokines which are secreted by virus-infected cellsas well as immune cells. The interferons function by binding to specificreceptors on cells adjacent to the infected cells, causing the change inthe cell which protects it from infection by the virus. α andβ-interferon also induce the expression of Class I and Class II MHCmolecules on the surface of infected cells, resulting in increasedantigen presentation for host immune cell recognition. α andβ-interferons are available as recombinant forms and have been used forthe treatment of chronic hepatitis B and C infection. At the dosageswhich are effective for anti-viral therapy, interferons have severe sideeffects such as fever, malaise and weight loss.

Immunoglobulin therapy is used for the prevention of viral infection.Immunoglobulin therapy for viral infections is different than bacterialinfections, because rather than being antigen-specific, theimmunoglobulin therapy functions by binding to extracellular virions andpreventing them from attaching to and entering cells which aresusceptible to the viral infection. The therapy is useful for theprevention of viral infection for the period of time that the antibodiesare present in the host. In general there are two types ofimmunoglobulin therapies, normal immunoglobulin therapy andhyper-immunoglobulin therapy. Normal immune globulin therapy utilizes aantibody product which is prepared from the serum of normal blood donorsand pooled. This pooled product contains low titers of antibody to awide range of human viruses, such as hepatitis A, parvovirus,enterovirus (especially in neonates). Hyper-immune globulin therapyutilizes antibodies which are prepared from the serum of individuals whohave high titers of an antibody to a particular virus. Those antibodiesare then used against a specific virus. Examples of hyper-immuneglobulins include zoster immune globulin (useful for the prevention ofvaricella in immuno-compromised children and neonates), human rabiesimmunoglobulin (useful in the post-exposure prophylaxis of a subjectbitten by a rabid animal), hepatitis B immune globulin (useful in theprevention of hepatitis B virus, especially in a subject exposed to thevirus), and RSV immune globulin (useful in the treatment of respiratorysyncitial virus infections).

Another type of immunoglobulin therapy is active immunization. Thisinvolves the administration of antibodies or antibody fragments to viralsurface proteins. Two types of vaccines which are available for activeimmunization of hepatitis B include serum-derived hepatitis B antibodiesand recombinant hepatitis B antibodies. Both are prepared from HBsAg.The antibodies are administered in three doses to subjects at high riskof infection with hepatitis B virus, such as health care workers, sexualpartners of chronic carriers, and infants.

Thus antiviral agents that can be combined with aziridino compounds inthe therapeutic compositions of the invention include nucleosideanalogs, normucleoside reverse transcriptase inhibitors, proteaseinhibitors, and integrase inhibitors. Specific examples of antiviralcompounds include the following: Acemannan; Acyclovir; Acyclovir Sodium;Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride;Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir;Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate;Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime;Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine;Fosarilate; Foscarnet Sodium; Fosfonet Sodium; Ganciclovir; GanciclovirSodium; Idoxuridine; Indinavir; Kethoxal; Lamivudine; Lobucavir;Memotine Hydrochloride; Methisazone; Nelfinavir; Nevirapine;Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; Ritonavir;Saquinavir Mesylate; Somantadine Hydrochloride; Sorivudine; Statolon;Stavudine; Tilorone Hydrochloride; Trifluridine; ValacyclovirHydrochloride; Vidarabine; Vidarabine Phosphate; Vidarabine SodiumPhosphate; Viroxime; Zalcitabine; Zidovudine; Zinviroxime and integraseinhibitors.

Parasiticides are agents that kill parasites directly. Such compoundsare known in the art and are generally commercially available. Examplesof parasiticides useful for human administration include but are notlimited to albendazole, amphotericin B, benznidazole, bithionol,chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine,diethylcarbamazine, diloxamide furoate, eflornithine, furazolidaone,glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole,mefloquine, meglumine antimoniate, melarsoprol, metrifonate,metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin,pentamidine isethionate, piperazine, praziquantel, primaquine phosphate,proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides,pyrimethanmine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidinegluconate, spiramycin, stibogluconate sodium (sodium antimonygluconate), suramin, tetracycline, doxycycline, thiabendazole,tinidazole, trimethroprim-sulfamethoxazole, and tryparsamide some ofwhich are used alone or in combination with others.

Parasiticides used in non-human subjects include piperazine,diethylcarbamazine, thiabendazole, fenbendazole, albendazole,oxfendazole, oxibendazole, febantel, levamisole, pyrantel tartrate,pyrantel pamoate, dichlorvos, ivermectin, doramectic, milbemycin oxime,iprinomectin, moxidectin, N-butyl chloride, toluene, hygromycin Bthiacetarsemide sodium, melarsomine, praziquantel, epsiprantel,benzimidazoles such as fenbendazole, albendazole, oxfendazole,clorsulon, albendazole, amprolium; decoquinate, lasalocid, monensinsulfadimethoxine; sulfamethazine, sulfaquinoxaline, metronidazole.

Parasiticides used in horses include mebendazole, oxfendazole, febantel,pyrantel, dichlorvos, trichlorfon, ivermectin, piperazine; for S.westeri: ivermectin, benzimiddazoles such as thiabendazole,cambendazole, oxibendazole and fenbendazole. Useful parasiticides indogs include milbemycin oxine, ivermectin, pyrantel pamoate and thecombination of ivermectin and pyrantel. The treatment of parasites inswine can include the use of levamisole, piperazine, pyrantel,thiabendazole, dichlorvos and fenbendazole. In sheep and goatsanthelmintic agents include levamisole or ivermectin. Caparsolate hasshown some efficacy in the treatment of D. immitis (heartworm) in cats.

Agents used in the prevention and treatment of protozoal diseases inpoultry, particularly trichomoniasis, can be administered in the feed orin the drinking water and include protozoacides such asaminonitrothiazole, dimetridazole (Emtryl), nithiazide (Hepzide) andEnheptin.

Anti-fungal agents are useful for the treatment and prevention ofinfective fungi. Anti-fungal agents are sometimes classified by theirmechanism of action. Some anti-fungal agents function as cell wallinhibitors by inhibiting glucose synthase. These include, but are notlimited to, basiungin/ECB. Other anti-fungal agents function bydestabilizing membrane integrity. These include, but are not limited to,imidazoles, such as clotrimazole, sertaconzole, fluconazole,itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,butenafine, and terbinafine. Other anti-fungal agents function bybreaking down chitin (e.g. chitinase) or immunosuppression (501 cream).

Some exemplary anti-fungal agents include imidazoles, FK 463,amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292, butenafine,chitinase and 501 cream, Acrisorcin; Ambruticin; Amorolfine,Amphotericin B; Azaconazole; Azaserine; Basifungin; Bifonazole;Biphenamine Hydrochloride; Bispyrithione Magsulfex; ButoconazoleNitrate; Calcium Undecylenate; Candicidin; Carbol-Fuchsin; Chlordantoin;Ciclopirox; Ciclopirox Olamine; Cilofungin; Cisconazole; Clotrimazole;Cuprimyxin; Denofungin; Dipyrithione; Doconazole; Econazole; EconazoleNitrate; Enilconazole; Ethonam Nitrate; Fenticonazole Nitrate; Filipin;Fluconazole; Flucytosine; Fungimycin; Griseofulvin; Hamycin;Isoconazole; Itraconazole; Kalafungin; Ketoconazole; Lomofungin;Lydimycin; Mepartricin; Miconazole; Miconazole Nitrate; Monensin;Monensin Sodium; Naftifine Hydrochloride; Neomycin Undecylenate;Nifuratel; Nifurmerone; Nitralamine Hydrochloride; Nystatin; OctanoicAcid; Orconazole Nitrate; Oxiconazole Nitrate; Oxifungin Hydrochloride;Parconazole Hydrochloride; Partricin; Potassium Iodide; Proclonol;Pyrithione Zinc; PyrroInitrin; Rutamycin; Sanguinarium Chloride;Saperconazole; Scopafungin; Selenium Sulfide; Sinefungin; SulconazoleNitrate; Terbinafine; Terconazole; Thiram; Ticlatone; Tioconazole;Tolciclate; Tolindate; Tolnaftate; Triacetin; Triafungin; UndecylenicAcid; Viridofulvin; Zinc Undecylenate; and Zinoconazole Hydrochloride.

The invention also provides combinations of two or more compounds thatinhibit cellular necrosis. The invention also provides combinations ofone or more compounds that inhibit cellular necrosis combined with oneor more additional agents or compounds (e.g., other therapeuticcompounds for treating a disease, condition, or infection).

The invention also provides kits including one or more compounds orcombinations of the invention (e.g., the heterocylic thiohydantoin,hydantoin, oxazolidinone, thioxo-oxazolidinone, pyrimidinone,oxazinanone compounds, or combinations thereof). A kit can also includeone or more additional agents or compounds described herein. Thedifferent components of the kit can be provided in different containers.The kit can be compartmentalized to receive the containers in closeconfinement. The kit can also contain instructions for using thecompounds according to the invention.

As used herein, a kit such as a compartmentalized kit includes any kitin which compounds or agents are contained in separate containers.Illustrative examples of such containers include, but are not limitedto, small glass containers, plastic containers or strips of plastic orpaper. Particularly preferred types of containers allow the skilledworker to efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers include, but are not limited to, a container that will accepta compound or combination of compounds and/or other agents of theinvention. One or more compounds or agents can be provided as a powder(e.g. lyophilized powder) or precipitate. Such compound(s) can beresuspended prior to administration in a solution that may be providedas part of the kit or separately available. A kit can contain compoundsor agents in other forms such as liquids, gels, solids, as describedherein. Different compounds and/or agents may be provided in differentforms in a single kit.

The term “ED₅₀” means the dose of a drug that produces 50% of itsmaximum response or effect. Alternatively, “ED₅₀” means the dose thatproduces a pre-determined response in 50% of test subjects orpreparations.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

The term “EC₅₀” means the concentration of a drug that produces 50% ofits maximum response or effect in a test assay. Alternatively, “EC₅₀”means the effective concentration that produces a pre-determinedresponse in 50% of test assays.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀-The term “structure-activity relationship (SAR)”refers to the way in which altering the molecular structure of drugsalters their interaction with a receptor, enzyme, etc.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

EXAMPLES Example 1 Screening Assays Used to Identify Inhibitors ofCellular Necrosis

After a cell receives an initial assault either apoptotic, necrotic, orboth apoptotic and necrotic mechanisms of cell death may be activated.The present example focuses on the necrosis pathway. Several chemicalassaults were used to induce cell death, including exposure to tumornecrosis factor alpha (TNF-α), Fas ligand or β-amyloid protein. Variouscell types were also used, including human neuroblastoma cells (SH-SY5Y)and human Jurkat T cells. In order to block the apoptosis mechanism, ageneral caspase inhibitor, N-benzyloxycarbonyl-valine-alanine-asparticacid-(OMe) fluoromethyl ketone (zVAD, Polyerino, A. J.; Patterson, S. D.J. Biol. Chem. 1997, 272, 7013-7021), was used. This compound inhibitsall caspases and consequently disrupts the apoptosis pathway. Resultingcell death occurs by a necrosis-like mechanism (Holler, N., et al.Nature Immunol. 2000, 1, 489-495; Kawahara, A., et al. J. Cell Biol.1998, 143, 1353-1360). Experimental compounds were applied to the cellsin attempts to rescue them from this necrotic death. Therefore,compounds found to restore cell viability using this protocol areinhibitors of the necrosis pathway.

Compound libraries were screened for inhibition of cell death induced byTNF-α in the presence of zVAD in human B cell line U-937. One compoundidentified as an inhibitor of necrosis was 1:

Compounds were also tested in another necrosis assay utilizing humanJurkat T cells, Fas ligand to induce cell death, and zVAD to inhibit theapoptosis pathway. After 36 h, cell viability was measure by thecommercial CellTiter ATP cell viability assay (Promega).

A structure-activity-relationships (SAR) study was conducted in order toincrease anti-necrosis activity. The compounds in Table 1 were preparedaccording to the procedures outlined in FIGS. 2 and 3. TABLE 1

Compound No. R₁ R₂ R₃ R₄ X Y 893-01 H H Me H S NH 893-02 H Me Me H S NH893-03 H H Me Me S NH 893-04 H H Et H O NH 893-05 6-F H Me H S NH 893-065-OMe H Me H S NH 893-07 5-OH H Me H S NH 893-08 H H Me H S NMe 893-097-F H Me H S NH 893-10 7-Cl H Me H S NH 893-11 6-Cl H Me H S NH 893-127-Br H Me H S NH 893-13 7-OMe H Me H S NH 893-14 5-Cl H Me H S S 893-157-Cl H Me H S NMe 893-16 6-SO₂Me; H Me H S NH 7-Cl 893-17 H H CH₂CH₂- HS NH morpholine 893-18 H H H H S NH 893-19 H H H H O NH 893-20 H H Me HO NH 893-21 H H Me H S S 893-22 H H Me H O NH 893-23 7-Me H Me H O NH893-24 5-Cl H Me H O NH 893-25 7-OMe H Me H O NH 893-26 5-OMe H Me H ONH 893-27 6-Cl H Me H O NH 893-28 7-F H Me H O NHMe = methyl, Et = ethyl

Other derivatives were also prepared utilizing similar procedures:

Compounds were screened for anti-necrotic activity utilizing humanJurkat T cells challenged with Fas ligand and treated with zVAD toinhibit the apoptosis pathway. Table 2 shows the EC₅₀ (μM) values ofselect compounds for cell viability. TABLE 2 Compound Number EC₅₀ (μM)893-01 6.0 893-04 10.0 893-05 2.3 893-08 35 893-09 4.0 893-10 1.5 893-1167.0 893-12 1.8 893-13 10.3 893-21 8.0 893-20 6.0

Compounds were screened for anti-necrotic activity utilizing humanFAD-deficient Jurkat T cells challenged with human TNF-alpha. FADD−/−Jurkat cells (Juo P, et al. Cell Growth Differ. 1999, 10(12):797-804)were seeded at the density of 5*10⁵ cells/mL into 96 well white plates(Costar) at 100 μL/well. Cells were treated in duplicate with variousconcentrations of test compounds in the presence or absence of 10 ng/mlhuman TNFα (Cell Sciences). After 30 hours viability of the cells wasdetermined using luminescent ATP-based cell viability assay(CellTiter-Glo, Promega). Percentage of protection by the compound wascalculated as a ratio of the cps (counts per second) value in the welltreated with the test compound and TNFα to the cps value in the welltreated with the compound alone. Table 3 shows the EC₅₀ (μM) values ofselect compounds for cell viability. TABLE 3 Compound Number EC₅₀ (μM)893-22 0.439 893-23 0.095 893-24 6.8 893-25 0.229 893-26 >300 893-271.12 893-28 0.324 893-31 0.303 893-32 0.078 893-33 >10 893-34 0.154893-35 0.448 893-36 >10 893-37 1.8 893-38 >10 893-39 5.4 893-40 >10893-41 >10 893-42 >10 893-43 >10 893-44 >10 893-45 >10 893-46 5.3893-47 >10 893-48 >10 893-49 4.3 893-50 >10 893-51 >10 893-52 >10 893-530.359 893-21 0.845 893-54 0.200

Inhibition of LPS-induced necrosis. RAW264.7 cells were maintained inRPMI1640 with antibiotic-antimycotic mixture and 10% FBS. One day priorto the experiment cells were seeded into 96 well plates at the densityof 5000 cells/well. Cells were treated with the indicated dose of LPSand 100 μM zVAD-fmk (marked “Z”, Q-Biogene), 0.25 μg/ml cyclohexamide(“C”, it potentiates aponecrosis induced by TNFalpha, Sigma) and 30 uMof compound 893-01. Cell viability was determined 24 hr later usingCellTiter-Glo ATP assay (Promega). Viability is expressed a percentageof the viable RAW264.7 macrophages in the treated well versus theuntreated control, which is set as 100% viability as shown in FIG. 1(cells treated with LPS and the apoptotic inhibitor zVAD (marked “Z” inFIG. 1) and/or cyclohexamide, a potentiator of aponecrosis (marked “C”in FIG. 1).

Accordingly, compounds of the invention are inhibitors of cellularnecrosis. The compounds are effective at maintaining cell viability whenthe cells were challenged with toxins (e.g. TNF-alpha, LPS) and theapoptosis pathway had been interrupted by the addition of zVAD. Thisprotection was found in different cell types, such as human neuronalcells, human T-cells and macrophages. Compounds described herein may beuseful as therapeutic agents (alone or in combination with othercompounds) for the treatment of humans afflicted with an acute orchronic disease. In addition, these compounds can be used in assaydevelopment of novel molecular targets integral to induced necrotic celldeath.

Example 2 Preparation of2-chloro-4-methanesulfonyl-6-trimethylsilanylethynyl-phenylamine

To a solution of 2-chloro-4-methanesulfonylaniline (822.6 mg, 4.0 mmol.)in dichloromethane (10 mL) were added, under argon,bis(pyridine)iodinium tetrafluoroborate (2.970 gm, 8 mmol.) andtrifluoromethanesulfonic acid (2.40 g, 16 mmol.). The reaction mixturestirred at room temperature for ˜16 h. It was diluted with water, andextracted in dichloromethane, dried and concentrated. The residue waspurified on the column using 0 to 40% ethyl acetate-hexane to give2-chloro-6-iodo-4-methanesulfonylaniline (969 mg, 73%): ¹H NMR (500 MHz,CDCl₃): 3.03 (s, 3H), 5.12 (s, 2H), 7.81 (s, 1H), 8.09 (s, 1H).

To the suspension of 2-chloro-6-iodo-4-methanesulfonylaniline (886.9 mg,2.7 mmol.), Pd(PPh₃)₂Cl₂ (94.5 mg, 0.13 mmol), and CuI (24.3 mg, 0.13mmol.) was added triethyl amine (2 mL), and the suspension was slowlytreated with (trimethylsilyl)acetylene (0.22 mL, 0.16 mmol.) at 0° C.The reaction mixture was stirred at room temperature for ˜16 h. Solventwas removed under vacuum. The residue was diluted with ethyl acetate,and filtered through Celite. The filtrate was washed with saturatedNaCl, water, dried over anhydrous sodium sulfate and concentrated. Theresidue was purified by column chromatography on silica gel using 20%ethyl acetate-hexane to give2-chloro-4-methanesulfonyl-6-trimethylsilanylethynyl-phenylamine (632mg, 78%). The ¹H NMR (500 MHz, CDCl₃) spectrum was: 0.27 (s, 9H), 3.01(s, 3H), 5.17 (s, 2H), 7.77 (d, J=2.5 Hz, 1H), 7.78 (d, J=2.5 Hz, 1H).

Example 3 Preparation of 7-Chloro-4-methanesulfonyl-1H-indole

To a mixture of2-chloro-4-methanesulfonyl-6-trimethylsilanylethynyl-phenylamine (100mg, 0.33 mmol.) and CuI (126.2 mg, 0.66 mmol.), DMF (2 mL) under argonwas added and the reaction mixture was heated at 100° C. for 2 hr. Thereaction mixture was diluted with ethyl acetate and filtered throughCelite. The filtrate was washed with saturated NaCl, dried andconcentrated. The residue was purified by column chromatography onsilica gel using 30% ethyl acetate-hexane to give7-Chloro-4-methanesulfonyl-1H-indole (38 mg, 50%): mp 160-162° C., ¹HNMR (500 MHz, CDCl₃): 3.09 (s, 3H), 6.75 (m, 1H), 7.43 (m, 1H), 7.75 (d,J=2.5 Hz, 1H), 8.19 (d, J=2.5 Hz, 1H), 8.84 (s, 1H).

Example 4 General Procedure for the Preparation of1H-indol-3-ylmethyl-dimethylamine, Exemplified for(7-Fluoro-1H-indol-3-ylmethyl)dimethyl-amine

To a mixture of acetic acid (13.6 mL) and formaldehyde (0.340 mL, 4.5mmol, 37% solution) under argon was added dimethyl amine (2.05 mL, 16.3mmol., 40% solution). The reaction mixture was stirred for 10 min andthen treated with 7-fluoroindole (540 mg, 4.0 mmol.). The resultingmixture was stirred at room temperature for ˜16 h. The reaction mixturewas first neutralized with K₂CO₃ and then basified with NaOH (2N), andthen extracted in ethyl acetate, washed with water, dried, andconcentrated. Solid obtained was recrystallized from ethyl acetate andhexane to give (7-Fluoro-1H-indol-3-ylmethyl)dimethylamine (570 mg,74%): mp 133-137° C., ¹H NMR (500 MHz, CDCl₃): 2.31 (s, 6H), 3.62 (s,2H), 6.88-6.92 (dd, J=8.0 and 8.0 Hz, 1H), 7.00-7.04 (m, 1H), 7.16 (d,J=2.5 Hz, 1H), 7.46 (d, J=8.0 Hz, 1H), 8.34 (s, 1H).

(7-Bromo-1H-indol-3-ylmethyl)dimethylamine: yield 81%, mp 113-118° C.,

¹H NMR (500 MHz, CDCl₃): 2.30 (s, 6H), 3.61 (s, 2H), 7.01 (dd, J=8.0 and8.0 Hz, 1H), 7.20 (d, J=2.0 Hz, 1H), 7.35 (d, J=7.5 Hz, 1H), 7.66 (d,J=8.0 Hz, 1H), 8.25 (s, 1H).

(7-Chloro-1H-indol-3-ylmethyl)dimethylamine: yield 86%, mp 136-138° C.,

¹H NMR (500 MHz, CDCl₃): 2.27(s, 6H), 3.61 (s, 2H), 7.04 (dd, J=8.0 and8.0 Hz, 1H), 7.15 (d, J=2.5 Hz, 1H), 7.18 (d, J=7.5 Hz, 1H), 7.60 (d,J=7.5 Hz, 1H), 8.53 (s, 1H).

(7-Methoxy-1H-indol-3-ylmethyl)dimethylamine: yield 81%, mp 99-102° C.,¹H NMR (500 MHz, CDCl₃): yield 81%, 2.27 (s, 6H), 3.62 (s, 2H), 3.95 (s,3H), 6.64 (d, J=7.5 Hz, 1H), 7.04 (dd, J=8.0 and 7.5 Hz, 1H), 7.11 (d,J=2.5 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H), 8.36 (s, 1H).

(7-Chloro-5-methanesulfonyl-1H-indol-3-ylmethyl)dimethylamine: yield82%, ¹H NMR (500 MHz, CDCl₃): 2.29 (s, 3H), 3.10 (s, 3H), 3.64 (s, 2H),7.37 (s, 1H), 7.77 (d, J=1.5 Hz, 1H), 8.30 (d, J=1.5 Hz, 1H), 8.63 (s,1H).

Example 5 General Procedure for the Preparation of2-(1H-indol-3-ylmethyl)-2-formylamino-malonic Acid Diethyl Esters.Preparation of 2-(7-Fluoro-1H-indol-3-ylmethyl)-2-formylamino-malonicAcid Diethyl Ester

A suspension of (7-fluoro-1H-indol-3-ylmethyl)-dimethyl-amine (550 mg,0.0028 mol.), 2-formylamino-malonic acid diethyl ester (640 mg, 0.0031mol.), and NaOH (30 mg) in toluene (20 mL) under argon was refluxed for3 days. The reaction mixture was concentrated and purified by columnchromatography on silica gel using 40% ethyl acetate-hexane to give2-(7-Fluoro-1H-indol-3-ylmethyl)-2-formylamino-malonic acid diethylester (1.0 gm, 99%): mp 164-166° C., ¹H NMR (500 MHz, CDCl₃): 1.28 (t,J=7.5 Hz, 6H), 3.87 (s, 2H), 4.17-4.31 (m, 4H), 6.80 (s, 1H), 6.86-6.89(dd, J=8.0 and 8.0 Hz, 1H), 6.97-7.01 (m, 2H), 7.27 (s, 1H), 8.18 (d,J=1.5 Hz, 1H), 8.27 (s, 1H).

2-(7-Bromo-1H-indol-3-ylmethyl)-2-formylamino-malonic acid diethylester: yield, 98%, mp 159-162° C., ¹H NMR (500 MHz, CDCl₃): 1.28 (t,J=7.5 Hz, 6H), 3.85 (s, 2H), 4.18-4.31 (m, 4H), 6.61 (d, J=8.0 Hz, 1H),6.78 (s, 1H), 6.90 (d, J=2.0 Hz, 1H), 6.99 (dd, J=7.5 and 8.0 Ha, 1H),7.10 (d, J=7.5 Hz, 1H), 8.18 (d, J=1.5 Hz, 1H), 8.28 (s, 1H).2-(7-Chloro-1H-indol-3-ylmethyl)-2-formylamino-malonic acid diethylester: yield 65%, mp 170-174° C., ¹H NMR (500 MHz, CDCl₃): 1.28 (t,J=7.5 Hz, 6H), 3.87 (s, 2H), 4.17-4.31 (m, 4H), 6.80 (s, 1H), 7.00 (d,J=2.5 Hz, 1H), 7.02 (dd, J=7.5 and 8.0 Hz, 1H), 7.17 (d, J=7.5 Hz, 1H),7.40 (d, J=8.0 Hz, 1H), 8.18 (d, J=1.0 Hz, 1H), 8.32 (s, 1H).

2-(7-Methoxy-1H-indol-3-ylmethyl)-2-formylamino-malonic acid diethylester: yield 73% (based on unrecovered starting compound), mp 149-153°C., ¹H NMR (500 MHz, CDCl₃): 1.28 (t, J=7.5 Hz, 6H), 3.85 (s, 2H), 3.94(s, 3H), 4.17-4.31 (m, 4H), 6.61 (d, J=8.0 Hz, 1H), 6.78 (s, 1H), 6.90(d, J=2.0 Hz, 1H), 6.99 (dd, J=7.5 and 7.5 Hz, 1H), 7.10 (d, J=7.5 Hz,1H), 8.18 (d, J=1.5 Hz, 1H), 8.28 (s, 1H).

2-(7-Chloro-5-methanesulfonyl-1H-indol-3-ylmethyl)-2-formylamino-malonicacid diethyl ester: yield 66%, mp 206-209° C., ¹H NMR (500 MHz, CDCl₃):1.15 (t, J=7.5 Hz, 6H), 3.21 (s, 3H), 3.67 (s, 2H), 4.09-4.17 (m, 4H),7.34 (s, 1H), 7.68 (d, J=1.0 Hz, 1H), 7.91 (d, J=1.5 Hz, 1H), 8.03 (s,1H), 8.68 (s, 1H), 12.07 (s, 1H).

Example 6 Preparation of2-(7-Chloro-1-methyl-1H-indol-3-ylmethyl)-2-formylamino-malonic AcidDiethyl Ester

To a suspension of DMSO (5 mL) and KOH (229 mg, 4.1 mmol) was added2-(7-Chloro-1H-indol-3-ylmethyl)-2-formylamino-malonic acid diethylester (500 mg, 1.4 mmol), followed by MeI (0.127 mL, 2 mmol) at 0° C.The reaction mixture was stirred for 4 hr. After the usual workup theproduct was purified by column chromatography on silica gel using 30%ethyl acetate—hexane to give2-(7-Chloro-1-methyl-1H-indol-3-ylmethyl)-2-formylamino-malonic aciddiethyl ester (402 mg, 77%): mp 83-87° C., ¹H NMR (500 MHz, CDCl₃): 1.28(t, J=7.5 Hz, 6H), 3.81 (s, 2H), 4.08 (s, 3H), 4.17-4.31 (m, 4H), 6.71(s, 1H), 6.93 (dd, J=7.5 and 7.5 Hz, 1H), 7.10 (d, J=7.5 Hz, 1H), 7.35(d, J=7.5 Hz, 1H), 8.18 (d, J=1.0 Hz, 1H).

Example 7 General Procedure for the Preparation of Tryptophans,Exemplified for DL-7-Fluoro-tryptophan

A solution of 2-(7-Fluoro-1H-indol-3-ylmethyl)-2-formylamino-malonicacid diethyl ester in THF was treated with NaOH (300 mg in 10 mL water)at room temperature for 24 hr. The mixture was slowly acidified withacetic acid (5 mL) and then refluxed for 24 hr. The reaction mixture wasconcentrated under vacuum, and treated with dil. HCl (10 mL, 3M) andthen again refluxed for 16 h. The reaction was allowed to cool to roomtemperature. The pH was adjusted to 6.0 with 2M KOH. The white solidthat formed was filtered, washed with water, and dried under vacuum togive 7-fluoro-tryptophan (282 mg, 52%): mp 256-261° C., ¹H NMR (500 MHz,DMSO-d₆): 2.92-2.97 (dd, J=8.5 and 15 Hz, 1H), 3.25-3.29 (dd, J=4.0 and15 Hz, 1H), 3.38-3.41 (dd, J=4.0 and 8.5 Hz, 1H), 6.87-6.96 (m, 2H),7.25 (s, 1H), 7.37 (d, J=7.5 Hz, 1H), 11.36 (s, 1H).

DL-7-Bromo-tryptophan: yield 92%, mp>260° C., ¹H NMR (500 MHz, DMSO-d₆):2.95-3.00 (dd, J=8.5 and 15 Hz, 1H), 3.25-3.29 (dd, J=4.0 and 15 Hz,1H), 3.40-3.42 (dd, J=4.0 and 8.5 Hz, 1H), 6.91 (dd, J=8.0 and 7.5 Hz,1H), 7.27 (d, J=2.0 Hz, 1H), 7.29 (d, J=7.5 Hz, 1H), 7.60 (d, J=8.0 Hz,1H), 11.11 (s, 1H).

DL-7-Chloro-tryptophan: yield 83%, mp 236-239° C., ¹H NMR (500 MHz,DMSO-d₆): 2.95-3.00 (dd, J=8.5 and 15 Hz, 1H), 3.24-3.28 (dd, J=4.0 and15 Hz, 1H), 3.41-3.43 (dd, J=4.0 and 8.5 Hz, 1H), 6.95 (dd, J=7.5 and7.5 Hz, 1H), 7.10 (d, J=7.5 Hz, 1H), 7.22 (s, 1H), 7.52 (d, J=7.5 Hz,1H), 11.18 (s, 1H).

DL-7-Chloro-N-methyl-tryptophan: yield 71%, mp 208-211° C., ¹H NMR (500MHz, DMSO-d₆): 2.95-3.43 (m, 3H) 4.02 (s, 3H), 6.92-6.96 (m, 1H), 7.09(d, J=7.5 Hz, 1H), 7.16 (s, 1H), 7.50-7.73 (dd, J=7.5 and 7.5 Hz, 1H).

DL-7-Methoxy-tryptophan: yield 46%, used as such for the next reactionwithout isolation.

DL-7-Chloro-5-methanesulfonyl-tryptophan: yield 83%, mp 292-294° C., ¹HNMR (500 MHz, DMSO-d₆): 3.04-3.08 (dd, J=8.5 and 15.5 Hz, 1H), 3.32 (m,1H), 3.44-3.47 (dd, J=4.5 and 8.5 Hz, 1H), 7.47 (s, 1H), 7.66 (d, J=1.5Hz, 1H), 8.20 (d, J=1.5 Hz, 1H), 11.90 (s, 1H).

Example 8 General Procedure for the Preparation of5-(1H-Indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-Ones fromTryptophan Esters, Exemplified for 893-01

To a solution of L-tryptophan methyl ester hydrochloride (0.254 mg,0.001 mol.) in dichloromethane (10 mL) was added triethyl amine (0.1 mL)followed by methylisothiocyanate (0.074 gm, 0.001 mol.). The reactionmixture was stirred at room temperature for 1 hr and then concentrated.The residue obtained was purified by column chromatography on silica gelusing 30% ethyl acetate in hexane to give 893-01 (230 mg, 89%): mp144-148° C., ¹H NMR (500 MHz, CDCl₃): 2.97-3.02 (dd, J=10.5 and 14.5 Hz,1H), 3.22 (s, 3H), 3.49-3.53 (dd, J=3.5 and 14.5 Hz, 1H), 4.36-4.39 (m,1H), 6.98 (s, 1H), 7.11 (d, J=2.5 Hz, 1H), 7.17 (t, J=8.0 Hz, 1H), 7.24(d, J=7.5 Hz, 1H), 7.40 (d, J=8.0 Hz, 1H), 7.60 (d, J=7.5 Hz, 1H), 8.16(s, 1H).

Example 9 General Procedure for the Preparation of5-(1H-Indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-ones fromTryptophans, Exemplified for 893-01

To a solution of L-tryptophan (0.408 gm, 0.002 mol.) in 50% aqueouspyridine (10 mL) was added methylisothiocyanate (0.175 gm, 0.0024 mol.)followed by the addition of NaOH (0.5 N) to pH (8-9). The reactionmixture was stirred at room temperature for 1 h and then extracted withpetroleum ether. Aqueous layer was acidified with concentrated HCl. Theacidic solution (pH 1.0) was left at room temperature for 2 days. Thenit was extracted in ethyl acetate, dried, and concentrated. The residueobtained was purified by column chromatography on silica gel using 30%ethyl acetate-hexane as an eluent to give 893-01 (42 mg, 8%): mp144-148° C.5-(1H-Indol-3-ylmethyl)-1,3-dimethyl-2-thioxo-imidazolidin-4-one(893-03):

Yield 15%, mp 132-135° C., ¹H NMR (400 MHz, CDCl₃): 3.04 (s, 3H), 3.22(s, 3H), 3.37-3.39 (m, 2H), 4.23-4.25 (m, 1H), 6.92 (d, J=1.6 Hz, 1H),7.08-7.18 (m, 2H), 7.31 (d, J=8.4 Hz, 1H), 7.55 (d, J=8.4 Hz, 1H), 8.04(s, 1H).

3-Methyl-5-(1-methyl-1H-Indol-3-ylmethyl)-2-thioxo-imidazolidin-4-one(893-08)

Yield 15%, mp 155-157° C., ¹H NMR (400 MHz, CDCl₃): 2.88-2.94 (dd,J=10.0 and 14.8 Hz, 1H), 3.45-3.50 (dd, J=4.0 and 14.8 Hz, 1H),4.30-4.34 (m, 1H), 6.81 (s, 1H), 6.92 (s, 1H), 7.10-7.31 (m, 3H), 7.54(d, J=8.0 Hz, 1H).

5-(1H-Indol-3-ylmethyl)-3,5-dimethyl-2-thioxo-imidazolidin-4-one(893-O₂)

Yield 57%, mp 179-182° C., ¹H NMR (400 MHz, CDCl₃): 1.42 (s, 3H), 3.10(s, 3H), 3.12-3.13 (m, 2H), 7.03 (s, 1H), 7.11-7.20 (m, 3H), 7.33 (d,J=7.2 Hz, 1H), 7.54 (d, J=8.0 Hz, 1H), 8.13 (s, 1H).

5-(6-Fluoro-1H-Indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-05)

Yield 24%, mp 126-129° C., ¹H NMR (400 MHz, CDCl₃): 2.94-3.00 (dd,J=10.0 and 14.8 Hz, 1H), 3.16 (s, 3H), 3.40-3.45 (dd, J=4.0 and 14.8 Hz,1H), 4.30-4.34 (m, 1H), 6.87-6.92 (m, 2H), 7.02-7.05 (m, 2H), 7.44-7.47(m, 1H), 8.09 (s, 1H).

5-(5-Methoxy-1H-Indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-06)

Yield 17%, mp 181-185° C., ¹H NMR (400 MHz, CDCl₃): 2.90-2.96 (dd,J=10.0 and 14.8 Hz, 1H), 3.19 (s, 3H), 3.41-3.46 (dd, J=4.0 and 14.8 Hz,1H), 3.83 (s, 3H), 4.30-4.34 (m, 1H), 6.85-6.87 (dd, J=2.4 and 8.4 Hz,1H), 6.91 (s, 3H), 6.96 (d, J=2.4 Hz, 1H), 7.04 (d, J=2.4 Hz, 1H), 7.25(d, J=8.4 Hz, 1H), 8.00 (s, 1H).

5-(5-Hydroxy-1H-Indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-07)

Yield 17%, mp 166-168° C., ¹H NMR (400 MHz, CDCl₃): 2.87-2.93 (dd,J=10.0 and 14.8 Hz, 1H), 3.18 (s, 3H), 3.37-3.41 (dd, J=4.0 and 14.8 Hz,1H), 4.27-4.31 (m, 1H), 6.76-6.79 (dd, J=2.4 and 8.4 Hz, 1H), 6.86 (s,3H), 6.94 (d, J=2.4 Hz, 1H), 7.04 (d, J=2.4 Hz, 1H), 7.23 (d, J=8.4 Hz,1H), 7.98 (s, 1H).

5-(7-Fluoro-1H-Indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-09)

Yield 31%, mp 217-220° C., ¹H NMR (500 MHz, CDCl₃): 3.00-3.05 (dd, J=9.5and 15.0 Hz, 1H), 3.22 (s, 3H), 3.47-3.51 (dd, J=4.0 and 15.0 Hz, 1H),4.36-4.39 (m, 1H), 6.83 (s, 1H), 6.94-6.98 (m, 1H), 7.06-7.10 (m, 1H),7.15 (d, J=2.0 Hz, 1H), 7.36 (d, J=8.0 Hz, 1H), 8.32 (s, 1H). Anal.Calcd for C₁₃H₁₂FlN₃OS: C, 56.30; H, 4.36; N, 15.15. Found: C, 56.12; H,4.39; N, 14.88.

5-(7-Bromo-1H-indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-12)

Yield 34%, mp 230-233° C., ¹H NMR (500 MHz, CDCl₃): 2.99-3.05 (dd, J=10and 15.0 Hz, 1H), 3.22 (s, 3H), 3.46-3.50 (dd, J=4.0 and 15.0 Hz, 1H),4.35-4.38 (m, 1H), 6.84 (s, 1H), 7.06 (t, J=7.5 Hz, 1H), 7.10 (d, J=2.5Hz, 1H), 7.40 (d, J=7.5 Hz, 1H), 7.54 (d, J=7.5 Hz, 1H), 8.32 (s, 1H).

5-(7-Chloro-1H-indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-10)

Yield 29%, mp 249-253° C., ¹H NMR (500 MHz, DMSO-d₆-CDCl₃): 3.02 (s,3H), 3.18-3.22 (dd, J=5.5 and 14.5 Hz, 1H), 3.30-3.34 (dd, J=4.5 and14.5 Hz, 1H), 4.35 (dd, J=4.5 and 5.5 Hz, H), 7.00 (t, J=7.5 Hz, 1H),7.13 (d, J=7.5 Hz, 1H), 7.19 (d, J=2.0 Hz, 1H), 7.52 (d, J=7.5 Hz, 1H),9.92 (s, 1H), 10.45 (s, 1H). Anal. Calcd for C₁₃H₁₂ClN₃OS: C, 53.15; H,4.12; N, 12.07. Found: C, 53.16; H, 4.21; N, 14.01.

5-(6-Chloro-1H-indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-11)

Yield 31%, ¹H NMR (500 MHz, DMSO-d₆): 3.13 (m, 1H), 3.30 (m, 1H), 3.32(s, 3H), 4.55-4.47 (m, 1H), 6.97-6.99 (dd, J=2.5 and 8.5 Hz, 1H), 7.19(s, 1H), 7.35 (d, J=2.5 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 10.33 (s, 1H),11.03 (s, 1H).

5-(7-Methoxy-1H-indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-13)

Yield 12%, mp 219-222° C., ¹H NMR (500 MHz, CDCl₃): 2.94-2.99 (dd, J=10and 15.0 Hz, 1H), 3.23 (s, 3H), 3.48-3.52 (dd, J=4.0 and 15.0 Hz, 1H),3.92 (s, 3H), 4.36-4.39 (m, 1H), 6.69 (d, J=7.5 Hz, 1H), 6.82 (s, 1H),7.07-7.11 (m, 2H), 7.19 (d, J=8.0 Hz, 1H), 8.33 (s, 1H).

5-(7-Chloro-1H-indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-15)

Yield 10%, mp 128-131° C., ¹H NMR (500 MHz, CDCl₃): 2.91-2.96 (dd, J=9.5and 15.5 Hz, 1H), 3.23 (s, 3H), 3.43-3.47 (dd, J=4.0 and 15.5 Hz, 1H),4.12 (s, 3H), 4.31-4.33 (m, 1H), 6.81 (s, 1H), 6.90 (s, 1H), 7.02 (dd,J=8.0 and 9.5 Hz, 1H), 7.18 (d, J=7.5 Hz, 1H), 7.44 (d, J=7.5 Hz, 1H).

5-(7-Chloro-5-methanesulfonyl-1H-indol-3-ylmethyl)-3-methyl-2-thioxo-imidazolidin-4-one(893-16)

Yield 26%, mp 242-245° C., ¹H NMR (500 MHz, CDCl₃): 3.12 (s, 3H), 3.13(s, 3H), 3.19-3.23 (dd, J=8.0 and 15.0 Hz, 1H), 3.42-3.46 (dd, J=4.0 and15.0 Hz, 1H), 4.41-4.44 (m, 1H), 7.11 (s, 1H), 7.33 (d, J=2.5 Hz, 1H),7.80 (s, J=1.0 Hz, 1H), 8.17 (d, J=1.0 Hz, 1H), 8.67 (s, 1H).3-Methyl-5-(1H-pyrrolo[2,3-b]pyridin-3-ylmethyl)-2-thioxo-imidazolidin-4-one (893-29):

Yield 41%, ¹H NMR (500 MHz, DMSO-d₆): 2.73 (s, 3H), 3.14-3.16 (m, 2H),4.55-4.58 (m, 1H), 7.16-7.32 (m, 2H), 8.24-8.32 (m, 2H), 10.29 (s, 1H),10.10 (s, 1H).

3-[Hydroxy-1-(methyl-5-oxo-2-thioxo-imidazolidin-4-yl)methyl]-indole-1-carboxylicAcid tert-butyl Ester (893-55)

Yield 18%, ¹H NMR (500 MHz, CDCl₃): 1.69 (s, 9H), 2.30 (m, 1H), 4.47 (m,1H), 5.37 (m, 1H), 6.69 (s, 1H), 7.26-7.41 (m, 2H), 7.59 (d, J=7.5 Hz,1H), 7.67 (s, 1H), 8.21 (d, J=8.5 Hz, 1H).

Example 10 Preparation of5-(1H-Indol-3-ylmethyl)-3-(2-morphlin-4-yl-ethyl)-2-thioxo-imidazolidin-4-one(893-17)

To a solution of L-tryptophan methyl ester hydrochloride (0.250 mg,0.001 mol.) in dichloromethane (10 mL) was added triethyl amine (3.0eq.) followed by 4-(2-isothiocyanato-ethyl)-morpholine (0.190 gm, 0.0011mol.). The reaction mixture was stirred at room temperature for 24 hr,and then concentrated. The residue obtained was purified by columnchromatography on silica gel using 60% ethyl acetate-hexane to give893-17 (292 mg, 83%): mp 167-169° C., ¹H NMR (500 MHz, CDCl₃): 2.45-2.51(m, 6H), 3.02-3.07 (dd, J=9.5 and 15.0 Hz, 1H), 3.46-3.50 (dd, J=4.0 and15.0 Hz, 1H), 3.65 (m, 4H) 3.89 (d, J=7.0 Hz, 2H), 4.35-4.38 (m, 1H),6.38 (s, 1H), 7.12 (d, J=2.5 Hz, 1H), 7.16-7.19 (m, 1H), 7.23-7.25 (dd,J=1.5 and 8.0 Hz, 1H), 7.39 (d, J=8.5 Hz, 1H), 7.61 (d, J=8.5 Hz, 1H),8.15 (s, 1H).

Example 11 Preparation of5-(1H-indol-3-ylmethyl)-2-thioxo-imidazolidin-4-one (893-18)

To a solution of L-tryptophan methyl ester hydrochloride (0.257 mg,0.001 mol.) in dichloromethane (10 mL) was added DMAP (5.0 mg),diisopropylethylamine (10 eq.) and then trimethylsilylisothiocyanate(1.3 gm, 0.01 mol.). The reaction mixture was stirred at roomtemperature for 24 hr, and then concentrated. The residue obtained wasdissolved in AcOH (10 mL) and refluxed for 6 h. The reaction mixture wasconcentrated and dried under vacuum. The solid obtained was suspended inEtOAc, filtered, washed with EtOAc, and dried under vacuum to give893-18 (230 mg, 94%): mp 206-210° C., ¹H NMR (500 MHz, DMSO-d₆):3.00-yield 34%, ¹H NMR (500 MHz, CDCl₃): 3.24-3.33 (m, 2H), 4.23-4.26(m, 1H), 7.01(dd, J=8.0 and 7.0 Hz, 1H), 7.09 (dd, J=8.0 and 7.0 Hz,1H), 7.23 (d, J=2.0 Hz, 1H), 7.37 (d, J=8.0 Hz, 1H), 7.50 (d, J=8.0 Hz,1H), 8.50 (s, 2H), 11.08 (s, 1H).

Example 12 Preparation of3-Ethyl-5-(1H-indol-3-ylmethyl)-imidazolidine-2,4-dione (893-04)

To a solution of L-tryptophan methyl ester hydrochloride (0.510 mg,0.002 mol.) in dichloromethane (10 mL) was added triethyl amine (0.350mL, 0.0025 mol.) followed by ethylisocyanate (0.198 mL, 0.0025 mol.).The reaction mixture was stirred at room temperature for 24 hr, and thenconcentrated. The residue obtained was diluted with dioxane (10 mL) andthen conc. HCl (5 mL) was added. The mixture was heated at reflux for3-4 hr. The reaction mixture was extracted in ethyl acetate, washed withwater, dried and concentrated. The residue was purified by columnchromatography on silica gel using 60% ethyl acetate-hexane to give893-04 (480 mg, 93%): mp 167-170° C. (lit. 163-164° C.), ¹H NMR (400MHz, CDCl₃): 0.99 (t, J=6.8 Hz, 3H), 2.95-3.01 (dd, J=8.4 and 14.8 Hz,1H), 3.34-3.46 (m, 3H), 4.19-4.22 (dd, J=3.6 and 8.8 Hz, 1H), 5.70 (s,1H), 6.98 (d, J=2.0 Hz, 1H), 7.06-7.10(dd, J=8.0 and 6.8 Hz, 1H),7.16(dd, J=7.2 and 6.8 Hz, 1H), 7.30 (d, J=8.4 Hz, 1H), 7.55 (d, J=8.8Hz, 1H), 8.18 (s, 1H).

Example 13 Preparation of5-(1H-indol-3-ylmethyl)-imidazolidine-2,4-dione (893-19)

To a solution of L-tryptophan methyl ester hydrochloride (0.500 mg,0.002 mol.) in dichloromethane (10 mL) was added triethyl amine (3.0 mL)followed by trimethylsilylisocyanate (2.300 gm, 0.02 mol.). The reactionmixture was stirred at room temperature for 24 hr and then concentrated.The residue obtained was dissolved in AcOH (5 mL) and refluxed for 5 hr.The reaction mixture was extracted in ethyl acetate, washed with water,dried and concentrated. The residue was dissolved in EtOH and treatedwith KOH, and stirred for 30 min. Then the reaction was concentrated anddried under vacuum to give5-(1H-indol-3-ylmethyl)-imidazolidine-2,4-dione potassium salt (245 mg,46%). This solid (50 mg, 0.18 mg) was acidified with dilute HCl at 0° C.The residue was extracted in ethyl acetate, and concentrated. The solidobtained after concentration was dried under vacuum to give 893-19 (42mg, 93%): mp 229-232° C., ¹H NMR (500 MHz, DMSO-d₆): 3.00-3.10 (m, 1H),3.23-3.27 (dd, J=4.0 and 14.5 Hz, 1H), 4.24-4.26 (m, 1H), 7.01(dd, J=7.5and 7.0 Hz, 1H), 7.09(dd, J=7.0 and 7.5 Hz, 1H), 7.13 (d, J=1.0 Hz, 1H),7.34-7.36 (m, 2H), 7.60 (d, J=8.0 Hz, 1H), 10.26 (s, 1H), 10.33 (s, 1H).

Example 14 Preparation of3-Methyl-5-(1H-indol-3-ylmethyl)-imidazolidine-2,4-dione (893-20)

5-(1H-indol-3-ylmethyl)-imidazolidine-2,4-dione potassium salt (200 mg,0.75 mmol.) obtained from above reaction was dissolved in DMF (5 mL),and treated with MeI (3 eq.). The reaction mixture was stirred at roomtemperature for 30 min., and then extracted with ethyl acetate, washedwith water, dried and concentrated. The solid obtained afterconcentration was recrystallized from ethyl acetate to give 893-20 (168mg, 92%): mp 207-210° C. (lit. mp 207-209° C.), ¹H NMR (500 MHz,CDCl₃-DMSO-d₆): 2.80 (s, 3H), 3.08-3.12 (dd, J=7.0 and 14.5 Hz, 1H),3.28-3.31 (dd, J=4.0 and 14.5 Hz, 1H), 4.25-4.28 (m, 1H), 7.00-7.10 (m,3H), 7.35 (d, J=7.5 Hz, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.79 (s, 1H), 10.39(s, 1H). Anal. Calcd for C₁₃H₁₃N₃O₂: C, 64.19; H, 5.39; N, 17.27. Found:C, 63.98; H, 5.44; N, 17.24.

Example 15 Preparation of5-(5-Chloro-benzo[b]thiophen-3-ylmethyl)-3-methyl-imidazolidine-2,4-dione(893-14)

To a mixture of 2-Amino-3-(5-chloro-benzo[b]thiophen-3-yl)-propionicacid (255.7 mg, 1.0 mmol) in methanol was added thionyl chloride (0.08mL) at 0° C. The resulting mixture was heated at reflux for 7 h, allowedto cool to room temperature, diluted with saturated sodium bicarbonateand extracted with ethyl acetate (2×30 mL). The organic extracts werecombined, washed with brine (25 mL), dried over anhydrous sodiumsulfate, filtered, and concentrated to give a yellow oil(2-amino-3-(5-chloro-benzo[b]thiophen-3-yl)-propionic acid methylester).

The oil (257 mg, 0.95 mmol) was dissolved in dichloromethane (20 mL) andthen methyl isothiocyanate (70 mg, 0.95 mmol) was added. The reactionmixture was heated at reflux for 24 h. The reaction mixture was allowedto cool to room temperature and then concentrated. The residue waspurified by column chromatography using hexane/ethyl acetate (75:25) aseluent to give a colorless oil(3-(5-chloro-benzo[b]thiophen-3-yl)-2-(3-methyl-thioureido)propionicacid methyl ester).

The oil (193 mg, 0.563 mmol) was stirred in dioxane (4 mL) withconcentrated HCl (2 mL) at 100° C. for 3 h. The mixture was allowed tocool to room temperature, diluted with water and extracted with ethylacetate. The organic layer was dried over anhydrous magnesium sulfate,filtered, and concentrated to give a yellow solid. The solid waspurified by column chromatography using hexane/ethyl acetate (80:20) aseluent to give 893-14 as a white solid: ¹H NMR (500 MHz, DMSO-d₆): 2.94(s, 3H), 3.23-3.32 (m, 2H), 4.66 (dt, J=1.0 and 5.5 Hz, 1H), 7.39 (dd,J=2.0 and 8.5 Hz, 1H), 7.56 (s, 1H), 7.35 (d, J=7.5 Hz, 1H), 7.92 (d,J=2.0 Hz, 1H), 8.01 (d, J=8.5 Hz, 1H), 10.96 (s, 1H).

Example 16 Procedure Used for the Preparation of 1-4. Exemplified forthe Preparation of 6-chloroindole-3-carboxaldehyde (1)

Phosphorus oxychloride (0.66 mL, 7 mmol) was added dropwise to anhydrousDMF (5 mL) at 0° C. under argon. A solution of 7-chloroindole (1 g, 6.6mmol) in anhydrous DMF (15 mL) was added dropwise at room temperatureand the resulting mixture was stirred for 2 h. The reaction mixture waspoured into ice and saturated NaHCO₃ and extracted with ethyl acetate.The combined organic solutions were washed with saturated NaCl (10mL×3), dried over anhydrous MgSO₄, filtered and concentrated to give 990mg of product, 1, as a yellow-orange solid (83%). ¹NMR (500 MHz,DMSO-d₆) δ 12.22 (1H, br s), 9.93 (1H, s), 8.34 (1H, s), 8.07 (1H, d,J=9.0 Hz), 7.57 (1H, d, J=1.5), 7.25 (1H, dd, J=1.8, 7.8 Hz).

(2)¹NMR (500 MHz, DMSO-d₆) δ 12.54 (1H, br s), 9.97 (1H, s), 8.39 (1H,s), 8.06 (1H, dd, J=1.3, 7.8 Hz), 7.37 (1H, dd, J=1.0, 7.5), 7.23 (1H,t, J=7.8 Hz,).

(3)¹NMR (500 MHz, DMSO-d₆) δ 12.32 (1H, br s), 9.92 (1H, s), 8.17 (1H,d, J=2.5), 7.66 (1H, d, J=8.0 Hz), 7.14 (1H, t, J=7.5 Hz), 6.84 (1H, d,J=7.5 Hz), 3.94 (3H, s).

(4)¹NMR (500 MHz, DMSO-d₆) δ 12.69 (1H, br s), 9.97 (1H, s), 8.37 (1H,s), 7.90 (1H, d, J=8.5 Hz), 7.20 (1H, dt, J=4.5, 8.0), 7.17 (1H, dd,J=7.5, 10.5 Hz).

Procedure used for the preparation of 5-12. Exemplified for thepreparation of5-(1H-Indol-3-ylmethylene)-3-methylimidazolidine-2,4-dione (5). Amixture of indole-3-carboxaldehyde (146 mg, 1 mmol) and1-methylimidazol-2,5(1,3H)-dione (synthesized according to the methodused in Eur. J. Org. Chem. 2002, 1763-1769) (250 mg, 2.5 mmol) inpiperidine (2 mL) was heated at 110° C. for 4 h under an argonatmosphere. Then the reaction mixture was allowed to cool in arefrigerator (˜5° C.) with the addition of ether (2 mL). The precipitatewas filtered and washed with ether to give 5 as a yellow solid (171 mg,71%). ¹NMR (500 MHz, DMSO-d₆): δ 11.84 (1H, br s), 10.29 (1H, br s),8.15 (1H, s), 7.79 (1H, d, J=7.5 Hz), 7.43 (1H, d, J=8.5 Hz), 7.18-7.12(1H, m), 7.13 (1H, td, J=1.0, 7.8), 6.86 (1H, s), 2.97 (3H, s).

Remark: For products that did not precipitate from reaction mixtures,ethyl acetate (200 mL) was added. The resulting solutions were washedsequentially with 1N HCl (50 mL×2), saturated NaHCO₃ (50 mL×2),saturated NaCl (50 mL), and then dried over anhydrous MgSO₄ with theaddition of 20 mL of MeOH. The mixtures were filtered and evaporated togives solids. Then 20 to 30% ethyl acetate in hexane solutions wasadded. The solid was filtered, washed with the same solvent to give theproducts as yellow solids. These solids were used without furtherpurification.

(6) ¹NMR (500 MHz, DMSO-d₆): δ 12.01 (1H, br s), 10.34 (1H, br s), 8.19(1H, s), 7.90 (1H, d, J=2.5 Hz), 7.44 (1H, d, J=9.0 Hz), 7.19 (1H, dd,J=2.0, 8.8 Hz), 6.85 (1H, s), 2.97 (3H, s).

(7) ¹NMR (500 MHz, DMSO-d₆): δ 11.95 (1H, br s), 10.37 (1H, br s), 8.17(1H, d, J=3.0), 7.83 (1H, d, J=9.0 Hz), 7.47 (1H, d, J=2.0 Hz), 7.13(1H, dd, J=2.0, 8.5 Hz), 6.83 (1H, s), 2.97 (3H, s).

(8) ¹NMR (500 MHz, DMSO-d₆): δ 12.15 (1H, br s), 10.26 (1H, br s), 8.23(1H, s), 7.79 (1H, d, J=8.0 Hz), 7.27 (1H, d, J=8.0 Hz,), 7.13 (1H, t,J=7.8 Hz), 6.82 (1H, s), 2.97 (3H, s).

(9) ¹NMR (500 MHz, DMSO-d₆): δ 11.80 (1H, br s), 10.35 (1H, br s), 8.16(1H, d, J=3.0), 7.60 (1H, d, J=8.0 Hz), 7.04 (1H, t, J=7.5 Hz), 6.99(1H, d, J=7.5 Hz), 6.84 (1H, s), 2.97 (3H, s), 2.48 (3H, s).

(10) ¹NMR (500 MHz, DMSO-d₆): δ 11.71 (1H, br s), 10.27 (1H, br s), 8.09(1H, s), 7.31 (1H, d, J=7.0 Hz), 7.30 (1H, s), 6.89 (1H, s), 6.81 (1H,dd, J=2.5, 8.5 Hz), 3.82 (3H, s), 2.97 (3H, s).

(11) ¹NMR (500 MHz, DMSO-d₆): δ 11.94 (1H, br s), 10.33 (1H, br s), 8.09(1H, d, J=3.0 Hz), 7.35 (1H, d, J=7.5 Hz), 7.06 (1H, t, J=7.8 Hz,), 6.81(1H, s), 6.76 (1H, d, J=7.5), 3.93 (3H, s), 2.97 (3H, s).

(12) ¹NMR (500 MHz, DMSO-d₆): δ 12.30 (1H, br s), 10.36 (1H, br s), 8.20(1H, s), 7.63 (1H, d, J=8.0 Hz), 7.10 (1H, dt, J=5.0, 8.0 Hz), 7.03 (1H,dd, J=7.5, 11.0 Hz), 6.82 (1H, s), 2.97 (3H, s).

Procedure used for the preparation of5-(1H-Indol-3-ylmethyl)-3-methylimidazolidine-2,4-diones. Exemplifiedfor the preparation of5-(1H-Indol-3-ylmethyl)-3-methylimidazolidine-2,4-dione (893-22). To asolution of 5-(1H-Indol-3-ylmethylene)-3-methylimidazolidine-2,4-dione(5) (120 mg, 0.5 mmol) in a mixed solvent of anhydrous MeOH/THF (1:1, 40mL) were added CoCl₂ (130 mg, 1.0 mmol) and NaBH₄ (380 mg, 10 mmol)portion wise. The mixture was stirred at room temperature overnight andthen diluted with ethyl acetate (100 mL). The mixture was washedsequentially with saturated NaHCO₃ (30 mL), 1N HCl (30 mL), saturatedNaCl (30 mL) and then dried over anhydrous MgSO₄, filtered andconcentrated. The crude product was purified by column chromatography onsilica gel using 50% ethyl acetate n hexane as eluent to give 893-22 asa white solid (80 mg, 66%). ¹H NMR (500 MHz, CDCl₃): δ 8.14 (1H, br s),7.61 (1H, d, J=7.0 Hz), 7.39 (1H, d, J=8.0 Hz), 7.22-7.25 (1H, m), 7.16(1H, t, J=7.8 Hz), 7.09 (1H, d, J=2.5), 5.32 (1H, br s), 4.30 (1H, ddd,J=1.0, 3.5, 10.0 Hz), 3.50 (1H, dd, J=4.0, 15.0 Hz), 2.99 (3H, s),2.94-2.97 (1H, m). (893-24) ¹H NMR (500 MHz, CDCl₃): δ 8.17 (1H, br s),7.58 (1H, d, J=2.0 Hz), 7.30 (1H, d, J=8.5 Hz), 7.18 (1H, dd, J=2.0, 8.5Hz), 7.12 (1H, d, J=2.5 Hz), 5.26 (1H, br s), 4.30 (1H, ddd, J=1.0, 3.7,9.4 Hz), 3.43 (1H, dd, J=3.8, 14.8 Hz), 2.99 (3H, s), 2.94-3.00 (1H, m).

(893-27) ¹H NMR (500 MHz, CDCl₃): δ 8.13 (1H, br s), 7.51 (1H, d, J=9.0Hz), 7.39 (1H, d, J=2.0 Hz), 7.13 (1H, dd, J=1.8, 8.8 Hz), 7.09 (1H, d,J=2.5 Hz), 5.20 (1H, br s), 4.28 (1H, ddd, J=2.5, 3.8, 9.0 Hz), 3.44(1H, dd, J=3.5, 15.0 Hz), 2.99 (1H, dd, J=9.0, 14.5 Hz), 2.97 (3H, s).

(893-54) ¹H NMR (500 MHz, CDCl₃): δ 8.43 (1H, br s), 7.50 (1H, d, J=8.0Hz), 7.22 (1H, d, J=7.5 Hz), 7.13 (1H, d, J=2.0 Hz), 7.06 (1H, t, J=7.8Hz), 5.69 (1H, br s), 4.27 (1H, ddd, J=1.0, 3.5, 8.8 Hz,), 3.43 (1H, dd,J=3.5, 14.5 Hz), 3.01 (1H, dd, J=9.3, 14.8 Hz), 2.95 (3H, s).

(893-23) ¹H NMR (500 MHz, CDCl₃): δ 8.08 (1H, br s), 7.46 (1H, d, J=8.0Hz), 7.03-7.10 (3H, m), 5.30 (1H, br s), 4.30 (1H, ddd, J=1.0, 3.9, 10.1Hz,), 3.48-3.52 (1H, m), 3.00 (3H, s), 2.95 (1H, dd, J=9.8, 14.8 Hz),2.50 (3H, s).

(893-26) ¹H NMR (500 MHz, CDCl₃): δ 8.06 (1H, br s), 7.27 (1H, d, J=8.5Hz), 7.05 (1H, d, J=2.5 Hz), 7.02 (1H, d, J=2.5 Hz), 6.89 (1H, dd,J=2.5, 8.5 Hz), 5.45 (1H, br s), 4.28-4.30 (1H, m), 3.86 (3H, s), 3.44(1H, dd, J=3.3, 14.8 Hz), 2.98 (3H, s), 2.94 (1H, dd, J=9.3, 14.8 Hz).

(893-25) ¹H NMR (500 MHz, CDCl₃): δ 8.33 (1H, br s), 7.21 (1H, d, J=8.0Hz), 7.08 (1H, t, J=7.3 Hz), 7.05 (1H, s), 6.68 (1H, d, J=7.5 Hz), 5.22(1H, br s), 4.29 (1H, dd, J=3.5, 10.0 Hz), 3.97 (3H, s), 3.49 (1H, dd,J=3.5, 14.5 Hz), 3.00 (3H, s), 2.93 (1H, dd, J=10.3, 14.8 Hz).

(893-28) ¹H NMR (500 MHz, CDCl₃): δ 8.35 (1H, br s), 7.36 (1H, d, J=7.5Hz), 7.11 (1H, d, J=2.0 Hz), 7.05 (1H, dt, J=4.5, 8.0 Hz), 6.93 (1H, dd,J=7.5, 10.5 Hz), 5.38 (1H, s), 4.29 (1H, dd, J=2.5, 9.0 Hz), 3.45 (1H,dd, J=3.5, 15.0 Hz), 2.99 (1H, dd, J=9.0, 15.0 Hz), 2.97 (3H, s).

Example 17 Preparation of Indoles

Several indoles needed were prepared utilizing the Bartoli synthesis(Bartoli, G.; Palmieri, G.; Bosco, M.; Dalpozzo R., Tetrahedron Lett.,1989, 2129). An example of this synthesis is illustrated for5,7-dichloroindole (FIG. 6).

A solution of 1.0 M vinylmagnesium bromide (45.0 mL, 45.0 mmol) wasquickly added to a stirred THF solution (30.0 mL) of2,4-dichloronitrobenzene (2.88 g, 15.0 mmol) cooled at −40° C. under N₂.The reaction mixture was stirred for 20 minutes and poured intosaturated aqueous ammonium chloride, extracted with ether and dried overanhydrous sodium sulfate. After chromatographic purification on silicagel, the indole was obtained in 46% yield (1.28 g). ¹H NMR (500 MHz,CDCl₃): δ 8.38 (1H, brs), 7.49 (1H, s), 7.52 (1H, d, J=1.5 Hz), 7.28(1H, t, J=8.0 Hz), 7.20 (1H, d, J=2.0 Hz), 6.54 (1H, dd, J=2.5, 3.5 Hz).

The known indole, 6,7-dichloroindole, and the previously unreportedindole, 7-chloro-2-methylindole (¹H NMR (500 MHz, CDCl₃): δ 8.07 (1H,brs), 7.39 (1H, d, J=7.5 Hz), 7.10 (1H, d, J=7.5 Hz), 6.99 (1H, t, J=8.0Hz), 6.26-6.24 (1H, m), 2.47 (3H, m) were prepared in a similar manner.

Example 18 Preparation of 7-oxygenated Indoles

7-Benzyloxyindole was prepared according to the published procedure ofHarada, et al. (Synthetic Communications 2003, 507). ¹H NMR (500 MHz,CDCl₃): δ 8.39 (1H, brs), 7.23 (1H, d, J=8.0 Hz), 7.17 (1H, t, J=3.0Hz), 7.01 (1H, t, J=7.5 Hz), 6.63 (1H, d, J=7.0 Hz), 6.53 (1H, t, J=2.5Hz), 4.14 (2H, t, J=6.5 Hz), 1.88-1.80 (2H, m), 1.59-1.50 (2H, m), 1.00(3H, t, J=7.0 Hz).

Furthermore, 7-benzyloxyindole (404 mg, 1.81 mmol) was hydrogenated over10% palladium on carbon (40 mg) in EtOH (4.2 mL) at ambient temperatureunder atmosphere pressure for 6 h. The catalyst was filtered off andwashed with EtOH. The filtrate was concentrated to give 7-hydroxyindoleas pale purple crystals, which was rapidly and directly used for thenext reaction.

To a stirred solution of 7-hydroxyindole and potassium carbonate (325mg, 2.35 mmol) in methyl ethyl ketone (2.4 mL) was added iodobutane(1.24 mL, 10.8 mmol) at room temperature. After the mixture was heatedat 55° C. for 12 h the solvent was removed and water was added. Themixture was extracted with EtOAc (3 times), dried over anhydrous MgSO₄,filtered and evaporated. After chromatographic purification on silicagel, 7-n-butoxyindole was obtained in 90% yield (310 mg). ¹H NMR (500MHz, CDCl₃) δ8.39 (1H, brs), 7.23 (1H, d, J=8.0 Hz, 7.17 (1H, t, J=3.0Hz), 7.01 (1H, t, J=7.5 Hz), 6.63 (1H, d, J=7.0 Hz), 6.53 (1H, t, J=2.5Hz), 4.14 (2H, t, J=5.2 Hz), 1.88-1.80 (2H, m), 1.59-1.50 (2H, m), 1.00(3H, t, J=7.0 Hz).

7-(2-Methoxy-ethoxy)-1H-indole was prepared in a similar as describedabove for the 7-benzyloxyindole.

The required 1-(2-methoxy-ethoxy)-3-methyl-2-nitrobenzene was preparedfrom 3-methyl-2-nitrophenyl. To a stirred solution of the phenyl (2.0 g,13.1 mmol) in DMF (65 mL) was added potassium carbonate (2.16 g, 15.7mmol) and bromoethyl methyl ether (1.47 mL, 15.7 mmol) at roomtemperature. After the mixture was stirred at 50° C. for 48 h, water wasadded. The mixture was extracted with EtOAc (3×), washed with brine,dried over anhydrous MgSO₄ filtered and evaporated. Afterchromatographic purification on silica gel,1-(2-methoxy-ethoxy)-3-methyl-2-nitrobenzene was obtained in 85% yield(2.34 g). ¹H NMR (500 MHz, CDCl₃) δ 7.28 (1H, t, J=8.0 Hz), 6.89 (1H, d,J=9.0 Hz), 6.85 (1H, d, J=8.0 Hz), 4.19 (2H, t, J=4.5 Hz), 3.72 (2H, t,J=5.0 Hz), 3.41 (3H, s), 2.30(3H, s).

Example 19 Preparation of (R)-7′-Chlorotryptophan, 1, and(S)-N-acetyl-7′-chlorotryptophan, 2

A mixture of N-acetyl-7′-chloro-DL-tryptophan (300 mg, 1.08 mmol),D-aminoacylase (10.1 MU/g, 8 mg) and cobalt dichloride (1.2 mg) in 30 mLof phosphate buffer solution (pH 7.8) was stirred at 37° C. for 2d. ThepH of the reaction mixture was adjusted to 5 with 10% HCl and thenfiltered through a celite pad. The filtrate was extracted with ethylacetate (3×40 mL). The aqueous layer was concentrated to give a paleyellow solid, which was extracted with methanol (4×2 mL). The combinedmethanol solutions were concentrated to give 200 mg of crude(R)-7′-chlorotryptophan as a white solid containing some inorganicsalts. This crude product was used directly for the next reactionwithout further purification. The ethyl acetate solutions were combinedand concentrated to give 150 mg of (S)-N-acetyl-7′-chlorotryptophan as alight yellow solid (100%).

(S)-7′-Chlorotryptophan, 3. Method A: (S)-N-acetyl-7′-chlorotryptophanwas refluxed in 3 N HCl for 6 h to remove the acetyl group.Concentration of the reaction mixture gave x mg of(S)-7′-chlorotryptophan (x %). Method B: Prepared with L-aminoacylaseusing the same procedure described for (R)-7′-chlorotryptophan.

(R)-7′-Chlorotryptophan methyl ester hydrochloride, 4. Thionyl chloride(0.09 mL, 1.2 mmol) was dissolved in 3 mL of anhydrous methanol at 0° C.and then this solution was added to a flask containing crude(R)-7′-chlorotryptophan (200 mg, 0.5 mmol). After stirring at −5° C. for4 h, the reaction mixture was allowed to warm to room temperature andstirred overnight before being concentrated. The white solid wascollected, washed with ethyl acetate and dried in vacuo. The product wasused directly without further purification.

(R)-7′-Chlorotryptophan methylamide hydrochloride, 5. To(R)-7′-chlorotryptophan methyl ester hydrochloride was added 4 mL of 2.0M solution of methyl amine in methanol. The mixture was stirred for 3dat room temperature under an atmosphere of argon. Concentration of thereaction mixture gave the crude product as a white solid, which was useddirectly without further purification.

(R)-5-(7′-Chloro-1H-indol-3ylmethyl)-3-methyl-imidazolidine-2,4-dione.To a mixture of the crude (R)-7′-chlorotryptophan methylamidehydrochloride (ca. 0.5 mmol), pyridine (0.24 mL, 3.0 mmol), anddichloromethane (6 mL) at 0° C. was slowly added triphosgene (178 mg,0.6 mmol) under argon. The reaction mixture was stirred at 0° C. for 1 hbefore removing the cooling bath. The stirring was continued overnightat room temperature under argon, then diluted with 120 mL of ethylacetate. The organic solution was washed with 1N HCl (2×40 mL) and brine(40 mL), dried over anhydrous MgSO₄, filtered and concentrated. Theresidue was purified by chromatography on silica gel using hexane/ethylacetate (50:50) to give 27 mg of pure product as a pale yellow solid(20% overall yield). ¹H NMR (500 MHz, CDCl₃): δ2.98 (s, 3H), 3.00 (dd,1H, J=9.0, 14.5 Hz), 3.46 (dd, 1H, J=3.5, 14.5 Hz), 4.29 (ddd, 1H,J=1.0, 3.5, 9.0 Hz), 5.30 (d, 1H, J=2.5 Hz), 7.09 (t, 1H, J=7.5 Hz),7.15 (d, 1H, J=2.5 Hz), 7.23 (d, 1H, J=7.5 Hz), 7.51 (d, 1H, J=7.5 Hz),8.35 (s, 1H). The optical purity (>98% ee) was determined by ¹H NMRusing a 0.02 M solution of the product in CDCl₃ in the presence of achiral shift reagent (Europiumtris[3-(trifluoromethylhydroxymethylene)-(+)-camphorate], 0.02 M).

(S)-5-(7′-Chloro-1H-indol-3ylmethyl)-3-methyl-imidazolidine-2,4-dione.Triphosgene (45 mg, 0.15 mmol) was added at 0° C. under argon to amixture of (S)-7′-chlorotryptophan methylamide hydrochloride (0.28mmol), pyridine (0.12 mL, 1.5 mmol), and dichloromethane (4 mL). Themixture was stirred at 0° C. for 2 h, then diluted with ethyl acetate(100 mL), washed with 1N HCl (2×30 mL) and brine (30 mL). The organiclayer was dried by MgSO₄, filtered and concentrated. The residue waspurified by chromatography on silica gel using dichloromethane/ethylacetate (85:15) to give 20 mg of pure product as a pale yellow solid(26%). ¹H NMR (500 MHz, CDCl₃): δ 2.98 (s, 3H), 3.00 (dd, 1H, J=9.0,14.5 Hz), 3.46 (dd, 1H, J=3.5, 14.5 Hz), 4.29 (ddd, 1H, J=1.0, 3.5, 9.0Hz), 5.30 (d, 1H, J=2.5 Hz), 7.09 (t, 1H, J=7.5 Hz), 7.15 (d, 1H, J=2.5Hz), 7.23 (d, 1H, J=7.5 Hz), 7.51 (d, 1H, J=7.5 Hz), 8.35 (s, 1H). Theoptical purity (>98% ee) was determined by ¹H NMR using a 0.02 Msolution of the product in CDCl₃ in the presence of a chiral shiftreagent (Europium tris[3(trifluoromethylhydroxymethylene)-(+)-camphorate], 0.02 M).

Example 20 Preparation of5-Benzo[b]thiophen-3-ylmethyl-3-methyl-2-thioxo-imidazolidin-4-one,893-21

To a solution of 2-amino-3-benzo[b]thiophen-3-yl-propionic acid (221 mg,1.0 mmol) in 4 mL pyridine/water (1:1) was added a solution ofmethylthioisocyanate (80.4 mg, 1.1 mmol). The resulting mixture wasstirred at 60° C. for 18 h. The reaction mixture was allowed to cool toroom temperature, diluted with 1N HCl (50 mL) and extracted with ethylacetate (2×40 mL). The extracts were combined, washed with brine, driedover anhydrous magnesium sulfate, filtered, and concentrated to give ayellow solid. The solid was dissolved in a mixture of ethylacetate/hexane/dichloromethane and then concentrated until precipitationbegan. The mixture was filtered to give 893-21 as a pale yellow solid.¹H NMR (500 MHz, CDCl₃): δ3.08 (dd, 1H, J₁=15 Hz, J₂=10 Hz), 3.26 (s,3H), 3.62 (dd, 1H, J₁=14.5 Hz, J₂=3.5 Hz), 4.42 (ddd, 1H, J=10 Hz, J₂=3Hz, J₃=1.0 Hz), 6.90 (bs, 1H), 7.30 (s, 1H), 7.40-7.46 (m, 2H), 7.77 (d,1H, J=8.0 Hz), 7.90 (d, 1H, J=8.0 Hz).

Example 21 Synthesis of Hydantoins

Methylhydantoin was prepared according to the method of Janin, Y. L., etal (Eur. J. Org. Chem., 2002, 1763). Ethyl and n-butylhydantoin wereprepared utilizing a literature procedure (Justus Liebigs Ann. Chem.,1903, 327 and 383). i-Propylhydantoin was prepared according to themethod of Park and Kurth (J. Org. Chem., 2000, 3520).

Example 22 Hydantoinindoles

The following hydantoinindoles were prepared utilizing the methodologydescribed in Example 16.

(893-33) ¹H NMR (500 MHz, CDCl₃): δ 8.35 (1H, brs), 7.49 (1H, s), 7.23(1H, s), 7.17 (1H, s), 5.43 (1H brs), 4.29 (1H, dd, J=3.5, 8.0 Hz), 3.38(1H, dd, J=4.0, 15.0 Hz), 3.01 (1H, dd, J=9.0, 15.0 Hz), 2.97 (3H, s).

(893-34) ¹H NMR (500 MHz, CDCl₃): δ 8.33 (1H, brs), 7.52 (1H, d J=8.0Hz), 7.23 (1H, d J=7.5 Hz), 7.15 (1H, d, J=2.5 Hz), 7.09 (1H, t, J=7.5Hz), 5.25 (1H brs), 4.28 (1H, ddd, J=1.0, 3.0, 8.0 Hz), 3.49 (2H, m),3.42 (1H, dd, J=3.0, 14.5 Hz), 3.04 (1H, dd, J=9.0, 14.5 Hz), 1.06 (3H,t, J=7.0 Hz).

(893-35) ¹H NMR (500 MHz, CDCl₃): δ 8.33 (1H, brs), 7.52 (1H, d J=7.5Hz), 7.22 (1H, d J=7.5 Hz), 7.15 (1H, d, J=2.5 Hz), 7.09 (1H, t, J=7.5Hz), 5.23 (1H brs), 4.28 (1H, ddd, J=1.5, 4.0, 8.0 Hz), 3.48-3.36 (3H,m), 3.07 (1H, dd, J=8.0, 14.5 Hz), 1.38 (2H, quintet, J=7.5 Hz),1.20-1.09 (2H, m), 0.84 (3H, t, J=7.0 Hz).

(893-38) ¹H NMR (500 MHz, CDCl₃): δ 8.08 (1H, brs), 7.46 (1H, d, J=8.0Hz), 7.11-7.02 (3H, m), 5.34 (1H, brs), 4.30 (1H, ddd, J=1.0, 4.0, 10.0Hz), 3.49 (1H, ddd, J=1.0, 4.0, 15.0 Hz), 3.00 (3H, s), 2.95 (1H, dd,J=9.5, 15.0 Hz), 2.49 (3H, s).

(893-43) ¹H NMR (500 MHz, CDCl₃): δ 8.13 (1H, brs), 7.39 (1H, d, J=10.0Hz), 7.15 (1H, d, J=9.0 Hz), 7.04 (1H, t, J=10.0 Hz), 5.20 (1H brs),4.25 (1H, ddd, J=1.0, 4.0, 9.0 Hz), 3.40 (1H, dd, J=4.5, 18.5 Hz), 3.00(s, 3H), 2.92 (1H, dd, J=12.5, 18.5 Hz), 2.45 (3H, s).

(893-44) ¹H NMR (500 MHz, CDCl₃): δ 8.33 (1H, brs), 7.51 (1H, d, J=8.0Hz), 7.22 (1H, d, J=7.0 Hz), 7.14 (1H, d, J=2.5 Hz), 7.08 (1H, t, J=8.0Hz), 5.32 (1H, brs), 4.24-4.15 (2H, m), 4.36 (1H, dd, J=9.0, 14.5 Hz),3.06 (1H, dd, J=8.5, 15.5 Hz), 1.26 (3H, d, J=7.0 Hz), 1.24 (3H, d,J=7.5 Hz).

(893-41) ¹H NMR (500 MHz, CDCl₃): δ 8.66 (1H, brs), 7.23 (1H, d, J=8.0Hz), 7.07-7.03 (2H, m), 6.70 (1H, d, J=7.5 Hz), 5.20 (1H, brs),4.32-4.26 (3H, m), 3.84-3.80 (2H, m), 3.48 (3H, s), 3.49 (1H, dd, J=3.0,15.0 Hz), 3.00 (3H, s), 2.93 (1H, dd, J=10.0, 15.0 Hz).

(893-0142) ¹H NMR (500 MHz, CDCl₃): δ 8.38 (1H, brs), 7.50-7.34 (5H, m),7.22 (1H, d, J=7.5 Hz), 7.06 (1H, d, J=8.0 Hz), 7.04 (1H, d, J=3.0 Hz),6.75 (1H, d, J=7.0 Hz), 5.25 (1H, brs), 5.21 (2H, s), 4.29 (1H, ddd,J=1.0, 3.5, 9.5 Hz), 3.48 (1H, dd, J=3.0, 14.5 Hz), 3.00 (3H, s), 2.93(1H, dd, J=10.5, 15.5 Hz).

(893-47) ¹H NMR (500 MHz, CDCl₃): δ 8.32 (1H, brs), 7.18 (1H, d, J=8.0Hz), 7.08-7.03 (2H, m), 6.66 (1H, d, J=8.0 Hz), 5.22 (1H, brs), 4.29(1H, ddd, J=1.0, 4.0, 9.5 Hz), 4.14 (2H, t, J=6.5 Hz), 3.48 (1H, dd,J=3.0, 14.0 Hz), 3.00 (3H, s), 2.93 (1H, dd, J=10.0, 14.5 Hz), 1.88-1.80(2H, m), 1.60-1.50 (2H, m), 1.00 (3H, t, J=7.5 Hz).

(893-50) ¹H NMR (500 MHz, CDCl₃): δ 8.33 (1H, brs), 7.43 (1H, d, J=8.0Hz), 7.22 (1H, d, J=8.0 Hz), 7.14 (1H, d, J=2.5 Hz), 5.19 (1H, brs),4.28 (1H, ddd, J=1.5, 4.0, 8.5 Hz), 3.40 (1H, dd, J=3.0, 14.5 Hz), 3.01(1H, dd, J=9.0, 15.0 Hz), 2.96 (3H, s).

Example 23 Synthesis of4-(7-Chloro-1H-indol-3-ylmethyl)-1-methyl-imidazolidin-2-one and5-(7-Chloro-1H-indol-3-ylmethyl)-3-methyl-oxazolidine-2,4-dione

The syntheses of4-(7-chloro-1H-indol-3-ylmethyl)-1-methyl-imidazolidin-2-one and5-(7-chloro-1H-indol-3-ylmethyl)-3-methyl-oxazolidine-2,4-dione wereaccomplished following the procedure of Lewis, R. et al. (J. Med. Chem.,1995, 923), except methyl amine was utilized in place of benzyl amines.

(893-52) ¹H NMR (500 MHz, CDCl₃): δ 8.33 (1H, brs), 7.48 (1H, d, J=9.5Hz), 7.22 (1H, d, J=9.0 Hz), 7.13 (1H, d, J=2.5 Hz), 7.08 (1H, t, J=10.0Hz), 4.40 (1H, brs), 3.95 (1H, quintet, J=8.5 Hz), 3.53 (1H, t, J=10.5Hz), 3.19 (1H, dd, J=6.5, 11.0 Hz), 2.98 (1H, dd, J=5.5, 17.5 Hz), 2.95(1H, dd, J=9.0, 18.0 Hz), 2.79 (3H, s).

(893-53) ¹H NMR (500 MHz, CDCl₃): δ 8.33 (1H, brs), 7.53 (1H, d, J=8.5Hz), 7.20 (1H, d, J=8.0 Hz), 7.19 (1H, d, J=2.5 Hz), 7.08 (1H, t, J=8.0Hz), 5.06 (1H, t, J=5.0 Hz), 3.48 (1H, dd, J=4.5, 15.5 Hz), 3.36 (1H,dd, J=5.5, 16.0 Hz) 2.84 (3H, s).

Example 24 Synthesis of4-(7-Chloro-1H-indol-3-ylmethyl)-oxazolidin-2-one

To a solution of 2 (500 mg, 1.78 mmol) in DMF solution (18 mL) was addedsodium bicarbonate (299 mg, 3.56 mmol) and iodomethane (0.554 mL, 8.9mmol). After the mixture was stirred at room temperature for 7 h, waterwas added. The mixture was extracted (3×) with ethyl acetate and theorganic layers were dried (MgSO₄) and evaporated to give ester as ayellow oil.

Lithium aluminum hydride (68 mg, 1.78 mmol) was suspended in ethersolution (15 mL). The ester in ether (3 mL) was added dropwise at 0° C.After stirred at room temperature for 1 h, the mixture was quenched withwater (0.068 mL) at 0° C., followed by addition of 15% NaOH solution(0.068 mL) and water (0.200 mL). The precipitate was filtered, theorganic filtrate was concentrated and the residue was purified bychromatography on silica gel to give alcohol 10 (370 mg, 78%).

A solution of the alcohol 10 (60 mg, 0.225 mmol) in 3N HCl solution washeated at 120° C. for 12 h. After cooled to room temperature, themixture was evaporated to give brown a solid, which was used for thenext reaction without purification.

The brown solid obtained above was dissolved in dichloromethane (2.25mL). Triethylamine (0.063 mL, 0.45 mmol) and 1,1′-carbonyldimidazole (73mg, 0.45 mmol) were added at room temperature. After stirred for 12 h,the mixture was concentrated and the residue was purified bychromatography on silica gel to give4-(7-chloro-1H-indol-3-ylmethyl)-oxazolidin-2-one, 11, as a white solid(24 mg, 42%). (893-51) ¹H NMR (500 MHz, CDCl₃): δ 8.35 (1H, brs), 7.47(1H, d, J=10.0 Hz), 7.24 (1H, d, J=10.0 Hz), 7.15 (1H, d, J=2.5 Hz),7.10 (1H, t, J=10.0 Hz), 5.14 (1H, brs), 4.56-4.44 (1H, m), 4.24-4.10(2H, m), 3.08-2.96 (2H, m).

Example 25 Synthesis of5-benzo[b]thiophen-3-ylmethyl-3-methyl-imidazolidine-2,4-dione

Thionyl chloride (0.36 mL, 4.8 mmol) was dissolved in 10 mL of anhydrousmethanol at −5° C. This solution was then added to a flask containingcrude (R)-3-benzothienylalanine (200 mg, 0.5 mmol). After stirring at−5° C. for 4 h, the reaction mixture was allowed to warm up overnightand then concentrated. The white solid[(R)-2-Amino-benzo[b]thiophen-2-yl-propionic acid methyl esterhydrochloride] was collected and washed with ethyl acetate. Thismaterial was then dried under vacuum and used directly for the nextstep.

To the crude (R)-2-Amino-benzo[b]thiophen-2-yl-propionic acid methylester hydrochloride was added 5 mL of 2.0 M solution of methyl amine inmethanol. The mixture was stirred for 2 d at room temperature underargon. Concentration of the mixture gave the crude product[(R)-2-Amino-benzo[b]thiophen-2-yl-N-methyl-propionamide hydrochloride]as a white solid (600 mg), which was used directly for the next reactionwithout further purification.

To a mixture of the crude(R)-2-Amino-benzo[b]thiophen-2-yl-N-methyl-propionamide hydrochloride(600 mg, ca. 2.0 mmol), triethylamine (0.6 mL, 4.0 mmol), anddichloromethane (20 mL) was added carbonyl diimidazole (2.44 g, 15mmol). The reaction mixture was stirred overnight at room temperatureunder argon and then diluted with 200 mL of ethyl acetate. The organicsolution was washed with 1N HCl (2×50 mL) and brine (60 mL), dried overanhydrous MgSO₄, filtered and concentrated. The residue was purified bycolumn chromatography on silica gel using hexane/ethyl acetate (50:50)to give 370 mg of5-benzo[b]thiophen-2-ylmethyl-3-methyl-imidazolidine-2,4-dione as awhite solid (71% overall yield). It is notable that the product wasracemic indicative that racemization had occurred under these reactionconditions. (893-39) ¹H NMR (500 MHz, CDCl₃): δ 7.89 (1H, dd, J=1.5, 7.5Hz), 7.78 (1H, dd, J=1.0, 7.0 Hz), 7.41 (2H, m), 7.27-7.24 (1H, m), 5.46(1H, s), 4.33 (1H, ddd, J=1.0, 3.5, 9.5 Hz), 3.60 (1H, ddd, J=1.0, 3.5,14.5 Hz), 3.03 (1H, dd, J=9.5, 14.5 Hz), 3.02(3H, s).

Example 26 Cytotoxicity of Hydantoin and Thiohydantoin Compound Series

FADD−/− Jurkat cells (Juo P, et al. Cell Growth Differ. 1999,10(12):797-804) were seeded at the density of 5*10⁵ cells/mL into 96well white plates (Costar) at 100 μL/well. Cells were treated induplicate with different concentrations of 893-10 or 893-54. After 30hours viability of the cells was determined using luminescent ATP-basedcell viability assay (CellTiter-Glo, Promega). Toxicity value wascalculated as a ratio of viable cells in the wells treated with thecompounds to the viable cells in the wells treated with DMSO (FIG. 11).

Example 27 Synthesis of5-(7-Chloro-1H-indol-3-ylmethyl)-1,3-dimethyl-imidazolidine-2,4-dione

To a solution of 1-methylhydantoin (1.14 g, 10 mmol) in methanol (40 mL)was added 10N NaOH (1 mL) and iodomethane (0.8 ml, 12.9 mmol). Themixture was refluxed for 4 h and then allowed to cool. The reactionmixture was diluted with EtOAc (200 mL), washed sequentially with 1N HCl(50 mL×3), saturated NaHCO₃ (50 mL×3), saturated NaCl (50 mL×3), driedover anhydrous MgSO₄, filtered and concentrated. The crude product(1,3-Dimethylimidazolidine-2,4-dione) as a slightly yellow oil was useddirectly for the next step without further purification.

5-(7-Chloro-1H-indol-3-ylmethylene)-1,3-dimethylimidazolidine-2,4-dionewas prepared 1,3-Dimethylimidazolidine-2,4-dione using the sameprocedure as described in Example 16. This material was used withoutfurther purification.

5-(7-Chloro-1H-indol-3-ylmethylene)-1,3-dimethylimidazolidine-2,4-dionewas converted to5-(7-Chloro-1H-indol-3-ylmethyl)-1,3-dimethylimidazolidine-2,4-dioneusing the same procedure as described in Example 16. (893-36) ¹H NMR(500 MHz, CDCl₃): δ 8.32 (1H, brs), 7.49 (1H, d J=8.0 Hz), 7.19 (1H, dJ=8.0 Hz), 7.08-7.04 (2H, m), 4.14 (1H, t, J=4.5 Hz), 3.37 (1H, dd,J=3.5, 15.0 Hz), 3.33 (1H, dd, J=5.0, 16.0 Hz), 2.93 (3H, s), 2.84 (3H,s).

Example 28 Synthesis of5-(7-Chloro-1-methyl-1H-indol-3-ylmethyl)-3-methyl-imidazolidine-2,4-dione

To a solution of 7-chloroindole (610 mg, 4 mmol) in anhydrous DMF (6 mL)was added NaH (170 mg, 60% dispersion in mineral oil, 4.3 mmol) at 0° C.under argon. The mixture was stirred at room temperature for 30 minbefore adding iodomethane (240 mg, 4 mmol). The resulting mixture wasstirred overnight. The reaction mixture was diluted with EtOAc (200 mL),washed with saturated NaCl (10 mL×3), dried over anhydrous MgSO₄,filtered, and concentrated to give 7-chloro-1-methylindole. The residuewas used directly for the next step without further purification. ¹H NMR(500 MHz, CDCl₃): δ 4.14 (s, 3H), 6.46 (d, 1H, J=2.5), 6.95-6.98 (m,2H), 7.12-7.14 (m, 1H), 7.48 (1H, dd, J=1.5, 7.5).

7-Chloro-1-methyl-1H-indole-3-carboxaldehyde was prepared from7-chloro-1-methylindole using the same procedure as described in Example16. ¹H NMR (500 MHz, CDCl₃): δ 4.23 (s, 3H), 7.19 (1H, t, J=7.5), 7.28(1H, dd, J=1.0, 7.5), 7.62 (s, 1H), 8.22-8.24 (1H, m), 9.98 (s, 1H).

5-(7-Chloro-1H-methyl-1H-indol-3-ylmethylene)-3-methylimidazolidine-2,4-dionewas prepared from 7-Chloro-1-methyl-1H-indole-3-carboxaldehyde using thesame procedure as described in Example 16. This crude material was usedin the next step without further purification.

5-(7-Chloro-1-methyl-1H-indol-3-ylmethyl)-3-methylimidazolidine-2,4-dionewas prepared from5-(7-Chloro-1-methyl-1H-indol-3-ylmethylene)-3-methylimidazolidine-2,4-dioneusing the same procedure as described in Example 16. (893-37) ¹H NMR(500 MHz, CDCl₃): δ 7.45 (1H, dd, J=1.0, 8.0 Hz), 7.17 (1H, dd, J=1.0,7.5 Hz), 7.01 (1H, t, J=7.5 Hz), 6.88 (1H, s), 5.25 (1H, brs), 4.24 (1H,ddd, J=1.0, 3.5, 9.0 Hz), 4.11 (3H, s), 3.42 (1H, dd, J=4.5, 15.0 Hz),2.99 (3H, s), 2.92 (1H, dd, J=10.0, 15.0 Hz).

Having thus described several aspects this invention, it is to beappreciated that various alterations, modifications, and improvementswill readily occur to those skilled in the art. Such alterations,modifications, and improvements are intended to be part of thisdisclosure, and are intended to be within the spirit and scope of theinvention. Accordingly, the foregoing description and drawings are byway of example only.

1. A compound of the formula:

a stereoisomeric form thereof, a pharmaceutically acceptable acid orbase addition salt thereof; wherein X represents O or S; Y represents Sor NR₈; G represents O or NR₇; R₁, R₂, and R₃ represent independently H,OH, OR₈, F, Cl, Br, I, N(R₈)₂, COOH, CO₂R₈, NO₂, NHC(O)R₈, lower alkyl,substituted lower alkyl, aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl; R₄ represents independently H, OH, OR₈, F, Cl,Br, I, N(R₈)₂, COOH, CO₂R₈, NO₂, NHC(O)R₈, methyl, methoxyl, loweralkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl, amine, piperizine; R₅, R₆, and R₇ representindependently H or lower alkyl; R₈ represents H, lower alkyl,substituted lower alkyl, aryl, substituted aryl, arylalkyl, alkenyl,alkynyl, heteroaryl, or substituted heteroaryl; R₉, R₁₀, R₉′, R₁₀′,represent independently H, F, Cl, Br, I, lower alkyl, substituted loweralkyl, or a three to six membered cycloalkyl or substituted cycloalkylthat includes C_(n) and/or C_(n)′; and n and n′ equals an integer fromzero to five; with the provision that when X is 0 and Y is NH, R₆ cannot be methyl, ethyl, propyl, isolpropyl or t-butyl when R₁, R₂, R₃, R₄,R₅ and R₇ are H.
 2. A pharmaceutical preparation comprising a compoundof claim 1 and a pharmaceutically acceptable carrier.
 3. Thepharmaceutical preparation of claim 2, wherein the pharmaceuticallyacceptable carrier is chosen from a diluent, a solid filler, and asolvent encapsulating material.
 4. The pharmaceutical preparation ofclaim 2, wherein the pharmaceutically acceptable carrier is chosen froma sugar, a starch, cellulose, powdered tragacanth, malt, gelatin, talc,an excipient, an oil, a glycol, a polyol, an ester, an agar, a bufferingagent, alginic acid, pyrogen free water, isotonic saline, Ringer'ssolution, ethyl alcohol, a pH buffered solution, a polyester, apolyanhydride, and a polycarbonate.
 5. A method of treating a necroticcell disease comprising administering to a subject having a necroticcell disease a compound of claim 1:

and wherein the compound is administered in an amount effective to treatthe necrotic cell disease.
 6. The method of claim 5, wherein thenecrotic cell disease is selected from the group consisting of trauma,ischemia, stroke, cardiac infarction, infection and sepsis.
 7. Themethod of claim 5, wherein the necrotic cell disease is aneurodegenerative disease chosen from Parkinson's disease, Alzheimer'sdisease, amyotrophic lateral sclerosis, Huntington's disease, andHIV-associated dementia.
 8. The method of claim 5, wherein the compoundis administered via a route chosen from oral, parenteral, topical,ocular, transdermal, and nasal routes.
 9. The method of claim 5, whereinthe compound is administered by subcutaneous, intramuscular,intravenous, or epidural injection.
 10. The method of claim 5, whereinthe compound is administered in combination with a pharmaceuticallyacceptable carrier.
 11. The method of claim 5, wherein the compound isadministered in combination with another compound.
 12. The method ofclaim 11, wherein the compound is selected from the list consisting ofapoptosis inhibitors, PARP inhibitors, Src inhibitors, agents fortreating stroke, agents for treating cardiovascular disorders andanti-microbial agents. 13-88. (canceled)
 89. A compound of the formula:

a stereoisomeric form thereof, a pharmaceutically acceptable acid orbase addition salt thereof, wherein X represents S or O; Y representsNH, or NR₈; G represents O or NR₇; R₁, R₂, and R₃ representindependently H, OR₈, F, Cl, Br, I, N(R₈)₂, CO₂R₈, NO₂, NHC(O)R₈, loweralkyl, substituted lower alkyl, aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl; R₄ represents independently H, OR₈, F, Cl, Br,I, N(R₈)₂, CO₂R₈, NO₂, NHC(O)R₈, aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl, amine, piperizine, lower alkyl and substitutedlower alkyl except for methyl and methoxyl; R₅, R₆ and R₇ representindependently H or lower alkyl; R₈ represents H, lower alkyl,substituted lower alkyl, aryl, substituted aryl, arylalkyl, alkenyl,alkynyl, heteroaryl, or substituted heteroaryl; R₉, R₁₀, R₉′, R₁₀′,represent independently H, F, Cl, Br, I, lower alkyl, substituted loweralkyl, or a three to six membered cycloalkyl or substituted cycloalkylthat includes C_(n) and/or C_(n)′; n and n′ equals an integer from zeroto five.
 90. The compound of claim 89, wherein R₄ represents a halogen,preferably Cl or F.
 91. The compound of claim 89, wherein R₄ representsCl, and wherein R₆ represents methyl.
 92. A pharmaceutical preparationcomprising a compound of claim 89 and a pharmaceutically acceptablecarrier. 93-94. (canceled)
 95. A method of treating a necrotic celldisease comprising administering to a subject having a necrotic celldisease a compound of claim 89:

wherein the compound is administered in an amount effective to treat thenecrotic cell disease.
 96. The method of claim 95, wherein R₄ representsa halogen, preferably Cl or F.
 97. The method of claim 95, wherein R₄represents Cl and wherein R₆ represents methyl.
 98. The method of claim95, wherein the necrotic cell disease is selected from the groupconsisting of trauma, ischemia, stroke, cardiac infarction, infectionand sepsis.